Bacillus combination for administration to animals

ABSTRACT

Embodiments of a composition comprising three or four  Bacillus  species selected from  Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis  and  Bacillus coagulans  are disclosed. The composition may further comprise, or be used in combination with, feed and/or feed supplements, including feed supplements comprising  yucca, quillaja , silica, mineral clay, glucan, mannans, or combinations thereof. Also disclosed is a method of administering the composition and/or combination to animals, such as poultry.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/930,577, filed on Jul. 16, 2020, which is a continuation ofInternational Application No. PCT/US2019/014938, filed Jan. 24, 2019,which was published in English under PCT Article 21(2), which in turnclaims the benefit of the earlier filing date of U.S. provisional patentapplication No. 62/621,196, filed on Jan. 24, 2018, all of which areincorporated herein by reference in their entireties.

FIELD

The present application concerns a combination and/or compositioncomprising three or four Bacillus species selected from Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus licheniformis andBacillus coagulans for administration to animals, particularly avians.

BACKGROUND

The gastrointestinal (GI) tracts of mammals are colonized by a diversecommunity of microflora. The GI tract may include hundreds of differentspecies, and this community profile may change over time based on ageand health of the individual. A healthy microbiota community in asubject provides many benefits, such as resistance to pathogens,nutrient absorption, and immune system performance. Intestinalmicrobiota also play a significant role in mediating pathogenicinfections of the gut, which significantly affect quality of life. Thegastrointestinal microflora compositions of both humans and animalssubstantially depend upon ingested materials. Accordingly, direct-fedmicrobial (DFM) compositions are commonly administered to influencephysiological health.

DFM products also are administered as an alternative to antibiotics inlivestock species. DFMs can restrict adherence of pathogenic microbes tomucosal surfaces, can stimulate an immune response or proliferation ofother endogenous beneficial microorganisms. Moreover, certain DFMsproduce and secrete other beneficial compounds or compositions, such asantimicrobial substances. Similar results between feeding DFM productsand prophylactic levels of antibiotics for growth have beendemonstrated. As a result, DFM products may be used as an alternative toantibiotics, or perhaps in combination with antibiotics.

Despite these prior DFM compositions, a need still exists for DFMcompositions that beneficially effect subjects receiving suchcompositions.

SUMMARY

Embodiments of a combination and/or composition comprising three or fourBacillus species selected from Bacillus amyloliquefaciens, Bacillussubtilis, Bacillus licheniformis and Bacillus coagulans are disclosed.The combination/composition is referred to herein as the Bacillicombination. In some embodiments, the composition comprises, consistsessentially of, or consists of, Bacillus subtilis, Bacilluslicheniformis and Bacillus amyloliquefaciens. In other embodiments, thecomposition comprises, consists essentially of, or consists of, Bacillussubtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, andBacillus coagulans. In further embodiments, the composition comprises,consists essentially of, or consists of, Bacillus coagulans, Bacillussubtilis, and Bacillus licheniformis.

Also disclosed are embodiments of an admixed composition comprising aBacilli combination and an additional component or composition.Exemplary admixed compositions comprise a first composition consistingessentially of, or consisting of, the Bacilli combination, and a secondcomponent or composition comprising one or more of a feed, yucca,quillaja, yucca and quillaja, a silica, mineral clay, a glucan andmannans mixture, a copper salt, a vitamin, an additional direct fedmicrobial, and any combination thereof.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table illustrating the results from the study described inExample 3.

FIG. 2 is a table illustrating results from the study described inExample 4.

FIG. 3 is a table illustrating additional results from the studydescribed in Example 4.

FIG. 4 is a table illustrating results from days 0-14 of the studydescribed in Example 6 for treatment groups T1-T11.

FIG. 5 is a table illustrating results from days 0-14 of the studydescribed in Example 6 for treatment groups T12-T15.

FIG. 6 is a table illustrating results from days 0-21 and 0-35 of thestudy described in Example 6 for treatment groups T1-T11.

FIG. 7 is a table illustrating results from days 0-21 and 0-35 of thestudy described in Example 6 for treatment groups T12-T15.

FIG. 8 is a table illustrating results from days 0-42 and 15-22 of thestudy described in Example 6 for treatment groups T1-T11.

FIG. 9 is a table illustrating results from days 0-42 and 15-22 of thestudy described in Example 6 for treatment groups T12-T15.

FIG. 10 is a table illustrating results from days 22-35 and 36-42 of thestudy described in Example 6 for treatment groups T1-T11, and alsoproviding the lesion scores.

FIG. 11 is a table illustrating results from days 22-35 and 36-42 of thestudy described in Example 6 for treatment groups T12-T15, and alsoproviding the lesion scores.

FIG. 12 is a table illustrating the intestinal bacterial data andprocessing data obtained from treatment groups T1-11 from the studydescribed in Example 6.

FIG. 13 is a table illustrating the intestinal bacterial data andprocessing data obtained from treatment groups T12-T15 from the studydescribed in Example 6.

FIG. 14 is a graph of live chicken body weights and feed:gain ratio,illustrating the effect of Magni-Phi® (250 ppm) alone, various DMFsalone, and Magni-Phi®/DFM combinations on body weight gain (0-14 days)and feed conversion (Feed:Gain 0-14 days) from the study described inExample 6, with the dotted lines indicating the polynomial trend linefor the respective graphs.

FIG. 15 is a graph of live chicken body weights and feed:gain ratio,illustrating the effect of Magni-Phi® (250 ppm) alone, various DMFsalone, and Magni-Phi®/DFM combinations on body weight gain (0-21 days)and feed conversion (Feed:Gain 0-21 days) from the study described inExample 6, with the dotted lines indicating the polynomial trend linefor the respective graphs.

FIG. 16 is a graph of live chicken body weights and feed:gain ratio,illustrating the effect of Magni-Phi® (250 ppm) alone, various DMFsalone, and Magni-Phi®/DFM combinations on body weight gain (0-35 days)and feed conversion (Feed:Gain 0-35 days) from the study described inExample 6, with the dotted lines indicating the polynomial trend linefor the respective graphs.

FIG. 17 is a graph of live chicken body weights and feed:gain ratio,illustrating the effect of Magni-Phi® (250 ppm) alone, various DMFsalone, and Magni-Phi®/DFM combinations on body weight gain (0-42 days)and feed conversion (Feed:Gain 0-42 days) from the study described inExample 6, with the dotted lines indicating the polynomial trend linefor the respective graphs.

FIG. 18 is a table of results from an in vitro culture test looking atpathogen growth inhibition.

FIG. 19 is a table of results from a second in vitro culture testlooking at pathogen growth inhibition.

DETAILED DESCRIPTION I. Terms

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Administering: Administration by any route to a subject, such aspoultry. In some embodiments, the route of administration is oral.

Antimicrobial: An agent that kills and/or inhibits the growth ofmicroorganisms. As used herein, antimicrobials include antibiotics,antifungals, antivirals, and antiparasitics including anticoccidials, orcombinations thereof.

Carrier: A substance that is used as an additive in (or with) acombination, composition, or component as disclosed herein. As usedherein, a carrier may be incorporated within particles of a combination,composition, or component, or it may be physically mixed with particlesof a combination, composition, or component. A carrier can be used, forexample, to modify non-biological properties of a combination orcomposition, such as flowability, stability during storage, exposure tomoisture, etc. Examples of carriers are included herein.

Colony forming units (CFU): “Colony forming units” refers to individualcolonies of bacteria. A colony is a mass of individual bacteria growingtogether. For certain embodiments, a colony comprises substantially thesame species, and may comprise, but does not necessarily comprise,substantially the same strain. CFU are a measure of the number ofbacteria present in or on a surface of a sample. However, CFU is notnecessarily a measure of individual cells or spores, as a colony may beformed from a single or a mass of cells or spores.

Combination: A combination includes two or more components that areadministered such that the effective time period of at least onecomponent overlaps with the effective time period of at least one othercomponent. A combination, or a component thereof, may be a composition.In some embodiments, effective time periods of all componentsadministered overlap with each other. In an exemplary embodiment of acombination comprising three components, the effective time period ofthe first component administered may overlap with the effective timeperiods of the second and third components, but the effective timeperiods of the second and third components independently may or may notoverlap with one another. In another exemplary embodiment of acombination comprising three components, the effective time period ofthe first component administered overlaps with the effective time periodof the second component, but not that of the third component; and theeffective time period of the second component overlaps with those of thefirst and third components. A combination may be a compositioncomprising the components, a composition comprising one or morecomponents and another separate component (or components) orcomposition(s) comprising the remaining component(s), or the combinationmay be two or more individual components. In some embodiments, the twoor more components may comprise the same component administered at twoor more different times, two or more different components administeredsubstantially simultaneously or sequentially in any order, or acombination thereof.

Bacilli Combination: Refers to a combination, or a composition, of DFMsincluding only three or four Bacillus species selected from Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus licheniformis andBacillus coagulans. In some disclosed embodiments, “Bacilli combination”refers to a composition for administration to a subject, particularly toan animal, and even more particularly to an avian, such as chickens andturkeys, that consists of or consists essentially of any three or fourof Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformisand Bacillus coagulans. In other embodiments, “Bacilli combination”refers to Bacillus amyloliquefaciens, Bacillus subtilis, Bacilluslicheniformis and Bacillus coagulans administered in combination withoutany other DFMs. A person of ordinary skill in the art will understandthat the Bacilli combination may include additional residual materialthat is carried over from the production of any or all of the three orfour Bacillus species, such as a dry milk product, and/or a carrier thatdoes not materially affect the structure, function, novel and/or basicfeatures of the Bacillus species.

CSL Combination: Refers to a combination, or a composition, of DFMsincluding only Bacillus coagulans, Bacillus subtilis and Bacilluslicheniformis. In some disclosed embodiments, “CSL combination” refersto a composition for administration to a subject, particularly to ananimal, and even more particularly to an avian, such as chickens andturkeys, that consists of or consists essentially of Bacillus coagulans,Bacillus subtilis and Bacillus licheniformis. In other embodiments, “CSLcombination” refers to Bacillus coagulans, Bacillus subtilis andBacillus licheniformis administered in combination without any otherDFMs. A person of ordinary skill in the art will understand that the CSLcombination may include additional residual material that is carriedover from the production of any or all of the three Bacillus species,such as a dry milk product, and/or a carrier that does not materiallyaffect the structure, function, novel and/or basic features of the threeBacillus species.

ASL Combination: Refers to a combination, or a composition, of DFMsincluding only Bacillus amyloliquefaciens, Bacillus subtilis andBacillus licheniformis. In some disclosed embodiments, “ASL combination”refers to a composition for administration to a subject, particularly toan animal, and even more particularly to an avian, such as chickens andturkeys, that consists of or consists essentially of Bacillusamyloliquefaciens, Bacillus subtilis and Bacillus licheniformis. Inother embodiments, “ASL combination” refers to Bacillusamyloliquefaciens, Bacillus subtilis and Bacillus licheniformisadministered in combination without any other DFMs. A person of ordinaryskill in the art will understand that the ASL combination may includeadditional residual material that is carried over from the production ofany or all of the three Bacillus species, such as a dry milk product,and/or a carrier that does not materially affect the structure,function, novel and/or basic features of the three Bacillus species.

ASLC Combination: Refers to a combination, or a composition, of DFMsincluding only Bacillus amyloliquefaciens, Bacillus subtilis, Bacilluslicheniformis and Bacillus coagulans. In some disclosed embodiments,“ASLC combination” refers to a composition for administration to asubject, particularly to an animal, and even more particularly to anavian, such as chickens and turkeys, that consists of or consistsessentially of Bacillus amyloliquefaciens, Bacillus subtilis, Bacilluslicheniformis and Bacillus coagulans. In other embodiments, “ASLCcombination” refers to Bacillus amyloliquefaciens, Bacillus subtilis,Bacillus licheniformis and Bacillus coagulans administered incombination without any other DFMs. A person of ordinary skill in theart will understand that the ASLC combination may include additionalresidual material that is carried over from the production of any or allof the four Bacillus species, such as a dry milk product, and/or acarrier that does not materially affect the structure, function, noveland/or basic features of the four Bacillus species.

Direct fed microbial: A composition that contains live and/or viablemicroorganisms, typically bacteria and/or yeast, that provides abeneficial effect on an animal, such as, but not limited to, anantimicrobial effect including decreased bacterial counts, improved feedconversion rate, improved weight gain, improved health parameters,reduced mortality rate, and/or improved lesion scores.

Feed conversion rate: A measure of the efficiency of an animal toconvert feed mass into increased body mass. Typically, the feedconversion rate is calculated as pounds of feed divided by pounds ofweight gain, and therefore may be expressed as a dimensionless number.The feed conversion rate is also known in the art as the feed conversionratio, or feed efficiency.

Mannans: A class of polysaccharides including the sugar mannose. Themannans family includes pure mannans (i.e., the polymer backbonecomprises of mannose monomers), glucomannan (the polymer backbonecomprises mannose and glucose), and galactomannan (mannans orglucomannan in which single galactose residues are linked to the polymerbackbone). Mannans are found in cell walls of some plant species andyeasts, and may be provided as extracts of such plant species and/oryeasts.

Mineral clay: The term “mineral clay” refers to hydrous aluminumsilicates. Mineral clays usually include minor amounts of impurities,such as potassium, sodium, calcium, magnesium, and/or iron.

Saponin: A class of chemical compounds, one of many secondarymetabolites found in natural sources. Saponins are found in particularabundance in various plant species, such as quillaja and yucca. Morespecifically, saponins are amphipathic glycosides grouped, in terms ofstructure, by their composition. In certain embodiments, a saponincomprises one or more hydrophilic glycoside moieties combined with alipophilic triterpene or a triterpene derivative, a steroid or asteroidal derivative, or both.

Strain: A strain refers to two members of the same species having adiscernible phenotypic and/or genetic difference.

Subject: Any animal or human, but particularly livestock (e.g., cows,sheep, goats, pigs, turkeys, and chickens) and household pets (e.g.,dogs, cats, and rodents), and most typically “subject” refers herein toavians, including poultry, such as chickens and turkeys.

Effective amount: A quantity or concentration of a specified compound,composition or combination sufficient to achieve an effect in a subject.

Vitamin: Includes Vitamin A, Vitamin B1 (thiamine), Vitamin B2(riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5(pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine,or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folicacid), Vitamin B12 (various cobalamins; commonly cyanocobalamin invitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 andK2 (i.e. MK-4, MK-7), folic acid and biotin, and derivative and analogsthereof.

Additional disclosure is provided by U.S. patent application Ser. No.14/699,740, U.S. patent application Ser. No. 13/566,433, U.S. patentapplication Ser. No. 13/872,935, U.S. Patent Publication No.2013/0017211, U.S. Patent Publication No. 2012/0156248, U.S. PatentPublication No. 2007/0253983, U.S. Patent Publication No. 2007/0202092,U.S. Patent Publication No. 2007/0238120, U.S. Patent Publication No.2006/0239992, U.S. Patent Publication No. 2005/0220846, U.S. PatentPublication No. 2005/0180964, and Australian Patent Application No.2011/201420, each of which is incorporated herein by reference in itsentirety.

II. Bacilli Combination

A Bacilli combination is a combination or composition comprising threeor four DFMs selected from Bacillus coagulans, Bacillus subtilis,Bacillus licheniformis and Bacillus amyloliquefaciens. A CSL combinationis a combination or composition comprising the DFMs Bacillus coagulans,Bacillus subtilis and Bacillus licheniformis and no additional DFMs. AnASL combination is a combination or composition comprising the DFMsBacillus amyloliquefaciens, Bacillus subtilis and Bacilluslicheniformis. In some embodiments, an ASL combination comprises,consists essentially of, or consists of Bacillus amyloliquefaciens,Bacillus subtilis and Bacillus licheniformis and no additional DFMs. AnASLC combination is a combination or composition comprising the DFMsBacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformisand Bacillus coagulans. In some embodiments, an ASLC combinationcomprises, consists essentially of, or consists of Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus licheniformis andBacillus coagulans, but no other additional DFMs.

Certain aspects of the present invention concern the discovery thatadministering a Bacilli combination, such as a CSL combination, an ASLcombination, or an ASLC combination, to a subject provides a substantialbenefit to the subject compared to a subject that is not administeredthe combination. The combination may be administered as a composition.With particular reference to poultry, a Bacilli combination provides asubstantial benefit with respect to one or more of feed conversion rate,average body weight, average body weight gain, body weight coefficientof variation, bird mortality, lesion scores, Salmonella/E.Coli/Clostridium perfingens (CP) incidence, and/or oocysts in fecalmatter relative to poultry fed none, one, or two of these bacilli in anycombination.

In some embodiments, one or more of the bacillus in the Bacillicombination is provided as a bacillus spore, and in certain embodiments,all of the bacillus in the Bacilli combination are provided as spores.

In some embodiments, one or more of the bacillus in the Bacillicombination is dehydrated, such as by freeze drying or lyophilization,spray drying or other suitable dehydration techniques. Dehydration, suchas by freeze drying or spray drying may improve the stability and/orshelf-life of the bacteria. In certain embodiments, all the bacillus inthe Bacillus combination are freeze dried.

A. Bacillus Strains

A person of ordinary skill in the art will appreciate that any strain,or combinations of strains, of Bacillus coagulans, Bacillus subtilis,Bacillus licheniformis and/or Bacillus amyloliquefaciens can be used inthe Bacilli combination. As used herein the terms “Bacillusamyloliquefaciens,” “Bacillus coagulans,” “Bacillus subtilis” and“Bacillus licheniformis” independently may refer to a single strain ofthe respective Bacillus species, or to multiple strains, such as 2, 3,4, 5, 6, 7, 8, 9, 10 or more strains, of each respective Bacillusspecies. Solely by way of example and without limitation, certainacceptable exemplary strains of each Bacillus species are listed below.In certain embodiments, a Bacillus combination includes one or more ofBacillus amyloliquefaciens TOA5001, Bacillus coagulans GBI-30 strain,ATCC Designation Number PTA-6086, Bacillus licheniformis OBT618, andBacillus subtilis strain OBT 1224, and in a particular embodiments, theBacillus combination includes Bacillus amyloliquefaciens TOA5001,Bacillus coagulans GBI-30 strain, ATCC Designation Number PTA-6086,Bacillus licheniformis OBT618, and Bacillus subtilis strain OBT 1224.

Bacillus coagulans Strains

Bacillus coagulans Hammer ATCC® BAA-738™ strain LMG 17453, Logan B0934,NCTC 3992, Vitek #202384, Bacillus coagulans Hammer ATCC® 7050™ strainNRS 609, NCIB 9365, NCTC 10334, DSM 1, CCM 2013, WDCM 00002, Bacilluscoagulans Hammer ATCC® 8038™ strain NCA 43P, NCIB 8080, NRS 770, DSM2312 deposited with ATCC as Bacillus thermoacidurans by Berry, Bacilluscoagulans Hammer ATCC® 10545™ strain NRS 784, NCIB 8041, DSM 2311, CCM1082, deposited with ATCC as Bacillus dextrolacticus by Andersen andWerkman, Bacillus coagulans Hammer ATCC® 11014™ strain NRS T27, 78G, DSM2383, Bacillus coagulans Hammer ATCC® 11369™ strain C, DSM 2384deposited with ATCC as Bacillus dextrolacticus by Andersen and Werkman,Bacillus coagulans Hammer ATCC® 12245™ strain NCA 308, DSM 2308, NCIB8870, Bacillus coagulans Hammer ATCC® 15949™ strain NCA 4259, DSM 2385,Bacillus coagulans Hammer ATCC® 23498™ strain M-39, DSM 2314, NCIB 10276deposited with ATCC as Bacillus racemilacticus by Nakayama and Yanoshi,Bacillus coagulans Hammer ATCC® 31284™ deposited with ATCC asLactobacillus sporogenes by Horowitz-Wiassowa and Nowotelnow, GanedenBiotech Inc.'s Bacillus coagulans GBI-30 strain, ATCC Designation NumberPTA-6086, Bacillus coagulans Hammer ATCC® 53595™ strain PM-1000,Bacillus coagulans Hammer strain DSM 2350, NRRL-NRS 2012, Bacilluscoagulans Hammer strain DSM 2356, NCIB 8523, N.R.Smith (NRS) 798, B.Hammer Iowa State College 200, Bacillus coagulans Hammer strain DSM30760, Bacillus coagulans Hammer strain STI09070 (IMET), 1032-005,Bacillus coagulans Hammer strain STI09076 (IMET), 1141-003, Bacilluscoagulans Hammer strain STI09080 (IMET), 1136-014, Bacillus coagulansHammer strain STI09208 (IMET), 491-25, Bacillus coagulans Hammer strainSTI09210 (IMET), 485-59, Bacillus coagulans Hammer strain NCIB 700460,Thl, Bacillus coagulans Hammer strain NCIB 701099, BG5, TH27 (205),Bacillus coagulans Hammer strain NCIB 701159, 254, and Bacilluscoagulans Hammer strain NCIB 701164, 259.

Bacillus licheniformis Strains

Bacillus licheniformis (Weigmann) Chester ATCC® 6598™ strain NRS 745deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn, Bacilluslicheniformis (Weigmann) Chester ATCC® 6634™ strain NRS 304, Bacilluslicheniformis (Weigmann) Chester ATCC® 8480™ strain NRS 1128, Bacilluslicheniformis (Weigmann) Chester ATCC® 9259™, Bacillus licheniformis(Weigmann) Chester ATCC® 9789™ strain AMNIH 723, ATCC 102, ATCC 4527,ATCC 8243, ATCC 9800, NCTC 2586, NCTC 6346, NRS 243, NRS 978, W. Ford 1,DSM 8785, DSM 46308, BU 171, CCDB b-30, CCEB 631, CCM 2205, CN 1060,HNCMB 101012, IFO 12195, IFO 12196, IMET 11025, NBRC 12195, NBRC 12196,NCDO 735, NCDO 835, NCIB 6346, NCIB 8059, NCIB 8061, OUT 8367, OUT 8368,Smith 243, Smith 978, HankeyB13 deposited with ATCC as Bacillus subtilisby (Ehrenberg) Cohn, Bacillus licheniformis (Weigmann) Chester ATCC®9945™ strain NRS 712, NCIB 8062 deposited with ATCC as Bacillus subtilisby (Ehrenberg) Cohn, Bacillus licheniformis (Weigmann) Chester ATCC®9945a™ strain CD-2, NCIB 11709, Bacillus licheniformis (Weigmann)Chester ATCC® 10716™ strain ATCC 11944, BS 2181, Boots 1343, CCM 2181,FDA BT1, NCIB 8874, NRS 1330, Tracy I, DSM 603, IFO 12199, NBRC 12199,Bacillus licheniformis (Weigmann) Chester ATCC® 11945™ strain 1331, FDABT3, Bacillus licheniformis (Weigmann) Chester ATCC® 11946™ strain 1333,B-1001, Bacillus licheniformis (Weigmann) Chester ATCC® 12139™ strainCSC deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn,Bacillus licheniformis (Weigmann) Chester ATCC® 12713™ strain PRL B479,NRRL B-1001, Bacillus licheniformis (Weigmann) Chester ATCC® 12759™strain ATCC 11560, Damodaron P-8, LMG 7560, NRS 1415, Vitek #200148,NCIB 8549, HankeyB133, P8, Bacillus licheniformis (Weigmann) ChesterATCC® 12759-MINI-PACK™ strain ATCC 11560, Damodaron P-8, LMG 7560, NRS1415, Vitek #200148, Bacillus licheniformis (Weigmann) Chester ATCC®13438™ Strain NCTC 8233, M. II strain, Bacillus licheniformis (Weigmann)Chester ATCC® 14409™ strain 620, NRS 1114, NCIB 1042, deposited withATCC as Bacillus abysseus by ZoBell and Upham, Bacillus licheniformis(Weigmann) Chester ATCC® 14580™ strain (Gibson) 46, NCIB 9375, NCTC10341, NRS 1264, DSM 13, CCM 2145, IFO 12200, NBRC 12200, WDCM 00068,Bacillus licheniformis (Weigmann) Chester ATCC® 14580D-5™ straindesignation: Genomic DNA from Bacillus licheniformis Strain 46 [ATCC®14580™], Bacillus licheniformis (Weigmann) Chester ATCC® 14594™,Bacillus licheniformis (Weigmann) Chester ATCC® 21038™ strain L-065,Bacillus licheniformis (Weigmann) Chester ATCC® 21039™, Bacilluslicheniformis (Weigmann) Chester ATCC® 21415™ strain NS 1 deposited withATCC as Bacillus subtilis by (Ehrenberg) Cohn, Bacillus licheniformis(Weigmann) Chester ATCC® 21417™ strain M deposited with ATCC as Bacillussubtilis (Ehrenberg) Cohn, Bacillus licheniformis (Weigmann) ChesterATCC® 21418™ deposited with ATCC as Bacillus subtilis by (Ehrenberg)Cohn, Bacillus licheniformis (Weigmann) Chester ATCC® 21424™ strain DSM1969, Bacillus licheniformis (Weigmann) Chester ATCC® 21610™ strainB-201-7 deposited with ATCC as Bacillus subtilis (Ehrenberg) Cohn,Bacillus licheniformis (Weigmann) Chester ATCC® 21667™ strain FD 23612,Bacillus licheniformis (Weigmann) Chester ATCC® 21733™ strain DSM 1913deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn, Bacilluslicheniformis (Weigmann) Chester ATCC® 25972™ strain 749/C, DSM 8782,DSM 46217, IMET10723, NCIB 9443, Bacillus licheniformis (Weigmann)Chester ATCC® 27326™ strain OM-81, Bacillus licheniformis (Weigmann)Chester ATCC® 27811™ strain 584, FERM-P 1038, Bacillus licheniformis(Weigmann) Chester ATCC® 31667™ strain DG 14, Bacillus licheniformis(Weigmann) Chester ATCC® 31972™ strain PM-3, Bacillus licheniformis(Weigmann) Chester ATCC® 33632™ strain (IOC) 2390, NCIB 11672, Bacilluslicheniformis (Weigmann) Chester ATCC® 39326™, Bacillus licheniformis(Weigmann) Chester ATCC® 53757™ strain PWD-1, Bacillus licheniformis(Weigmann) Chester ATCC® 53926™ strain E312, Bacillus licheniformis(Weigmann) Chester ATCC® 55768™ strain O.W.U. 138B [OWU 138B], Bacilluslicheniformis (Weigmann) Chester strain DSM 15, C, Bacilluslicheniformis (Weigmann) Chester strain DSM 392, Bacillus licheniformis(Weigmann) Chester strain DSM 394, Bacillus licheniformis (Weigmann)Chester strain DSM 7259, NRRL-NRS 1263, Bacillus licheniformis(Weigmann) Chester strain DSM 7459, Bacillus licheniformis (Weigmann)Chester strain DSM 11258, Bacillus licheniformis (Weigmann) Chesterstrain DSM 11259, Bacillus licheniformis (Weigmann) Chester strain DSM12369, Bacillus licheniformis (Weigmann) Chester strain DSM 12370,Bacillus licheniformis (Weigmann) Chester strain DSM 26543, Bacilluslicheniformis (Weigmann) Chester strain DSM 28096, Bacilluslicheniformis (Weigmann) Chester strain DSM 28591, Bacilluslicheniformis (Weigmann) Chester strain DSM 30523, Bacilluslicheniformis (Weigmann) Chester strain DSM 30535, Bacilluslicheniformis (Weigmann) Chester strain DSM 30542, Bacilluslicheniformis (Weigmann) Chester strain DSM 30585, Bacilluslicheniformis (Weigmann) Chester strain DSM 30615, Bacilluslicheniformis (Weigmann) Chester strain DSM 30620, Bacilluslicheniformis (Weigmann) Chester strain DSM 30624, Bacilluslicheniformis (Weigmann) Chester strain DSM 30643, Bacilluslicheniformis (Weigmann) Chester strain DSM 30654, Bacilluslicheniformis (Weigmann) Chester strain DSM 30724, Bacilluslicheniformis (Weigmann) Chester strain DSM 30766, Bacilluslicheniformis (Weigmann) Chester strain DSM 30769, Bacilluslicheniformis (Weigmann) Chester strain DSM 30778, Bacilluslicheniformis (Weigmann) Chester strain DSM 30779, Bacilluslicheniformis (Weigmann) Chester strain DSM 30865, Bacilluslicheniformis (Weigmann) Chester strain DSM 30926, Bacilluslicheniformis (Weigmann) Chester strain DSM 30959, Bacilluslicheniformis (Weigmann) Chester strain DSM 30960, Bacilluslicheniformis (Weigmann) Chester strain DSM 30961, Bacilluslicheniformis (Weigmann) Chester strain DSM 30976, Bacilluslicheniformis (Weigmann) Chester strain DSM 31019, Bacilluslicheniformis (Weigmann) Chester strain DSM 100653, Bacilluslicheniformis (Weigmann) Chester strain DSM 100655, Bacilluslicheniformis (Weigmann) Chester strain DSM 103059, Bacilluslicheniformis (Weigmann) Chester strain NCIB 1525, 1229, Bacilluslicheniformis (Weigmann) Chester strain NCIB 6816, Glaxo 417, Bacilluslicheniformis (Weigmann) Chester strain NCIB 7224, Loos, Bacilluslicheniformis (Weigmann) Chester strain NCIB 8536, P1, Bacilluslicheniformis (Weigmann) Chester strain NCIB 8537, Ho, Bacilluslicheniformis (Weigmann) Chester strain NCIB 9536, Gibson 1319, NRS1553, Bacillus licheniformis (Weigmann) Chester strain NCIB 9667, 1,Bacillus licheniformis (Weigmann) Chester strain NCIB 9668, 2, Bacilluslicheniformis (Weigmann) Chester strain NCIB 9669, 3, Bacilluslicheniformis (Weigmann) Chester strain NCIB 10689, Bacilluslicheniformis (Weigmann) Chester strain NCIB 11143, Bacilluslicheniformis (Weigmann) Chester strain NCIB 11643, YNS7712R, Bacilluslicheniformis (Weigmann) Chester strain NCIB 13497, Bacilluslicheniformis (Weigmann) Chester strain NCIB 14014, DA33, Bacilluslicheniformis B1 (NRRL Deposit Number B-50907), Bacillus subtilis B2(Deposit Number B-50908), Bacillus licheniformis RW25 (NRRL DepositNumber B-50911), Bacillus licheniformis RW32 (NRRL Deposit NumberB-50912), and Bacillus licheniformis RW41 (NRRL Deposit Number B-50913),Bacillus lichemformis BL21 (NRRL B-50134), Bacillus lichemformis 3-12a(NRRL B-50504), Bacillus licheniformis 4-2a (NRRL B-50506), Bacilluslicheniformis 842 (NRRL B-50516), Bacillus lichemformis DSM 5749(BioPlus® 2B, Chr. Hansen Bio Systems), and Bacillus licheniformisOBT618 (ATCC PTA-122188, Osprey Biotechnics).

Bacillus subtilis Strains

Bacillus subtilis (Ehrenberg) Cohn ATCC®82™ strain AMC, ATCC 8037, NRS315, Bacillus subtilis (Ehrenberg) Cohn ATCC©82D-5™ strain designation:Genomic DNA from Bacillus subtilis strain AMC [ATCC® 82™], Bacillussubtilis (Ehrenberg) Cohn ATCC®465™ strain NRS 743, Bacillus subtilis(Ehrenberg) Cohn ATCC®4529™ strain 3, ATCC 8013, NCTC 2588, NRS 1004deposited with ATCC as Bacillus vulgatus by Trevisan, Bacillus subtilis(Ehrenberg) Cohn ATCC®4925™ strain NRS 740 deposited with ATCC asBacillus nigrificans by Fabian and Nienhuis, Bacillus subtilis(Ehrenberg) Cohn ATCC®4944™ strain NCTC, NRS 1106 deposited with ATCC asBacillus parvus, Bacillus subtilis subspecies subtilis (Ehrenberg) CohnATCC® 6051™ strain Marburg strain, ATCC 6051-U, CCM 2216, CCRC 10255,CCUG 163B, CFBP 4228, CIP 52.65, DSM 10, IAM 12118, IFO 12210, IFO13719, IFO 16412, IMET 10758, JCM 1465, LMG 7135, NCAIM B.01095, NCCB32009, NCCB 53016, NCCB 70064, NCFB 1769, NCIB 3610, NCTC 3610, NRRLB-4219, NRS 1315, NRS 744, VKM B-501, NBRC 13719 deposited with ATCC asBacillus subtilis (Ehrenberg) Cohn, Bacillus subtilis (Ehrenberg) CohnATCC®6051a™ strain P31K6, Bacillus subtilis bacteriophage phi-eATCC®6051-B1™ strain Phi-e deposited with ATCC as phi e, Bacillussubtilis (Ehrenberg) Cohn ATCC©6460™ strain NRS 259 deposited with ATCCas Bacillus aterrimus by Lehmann and Neumann, Bacillus subtilis(Ehrenberg) Cohn ATCC©6461™ strain NRS 275, CN 2192, NCIB 8055 depositedwith ATCC as Bacillus aterrimus by Lehmann and Neumann, Bacillussubtilis subspecies spizizenii Nakamura et al. ATCC® 6633™ strain NRS231, DSM 347, CCM 1999, IAM 1069, NCIB 8054, NCTC 10400, WDCM 00003deposited with ATCC as Bacillus subtilis (Ehrenberg) Cohn, Bacillussubtilis subspecies spizizenii Nakamura et al. ATCC® 6633D-5™ straindesignation: Genomic DNA from Bacillus subtilis subspecies spizizeniistrain NRS 231 [ATCC®6633™] deposited with ATCC as Bacillus subtilis(Ehrenberg) Cohn, Bacillus subtilis subspecies spizizenii Nakamura etal. ATCC® CRM-6633™ strain NRS 231 deposited with ATCC as Bacillussubtilis (Ehrenberg) Cohn, Bacillus subtilis subspecies spizizeniiNakamura et al. ATCC® 6633-MINI-PACK™ strain NRS 231 deposited with ATCCas Bacillus subtilis (Ehrenberg) Cohn, Bacillus subtilis (Ehrenberg)Cohn ATCC©6984™ strain NRS 747 deposited with ATCC as Bacillus vulgatussubspecies hydrolyticus, Bacillus subtilis (Ehrenberg) Cohn ATCC®7003™strain NRS 730, Bacillus subtilis (Ehrenberg) Cohn ATCC®7058™ strain NRS351, Bacillus subtilis (Ehrenberg) Cohn ATCC®7059™ strain NRS 352,Bacillus subtilis (Ehrenberg) Cohn ATCC®7060™ strain NRS 659, Bacillussubtilis (Ehrenberg) Cohn ATCC®7067™ strain NRS 238, ATCC 7974, ATCC8012, Bacillus subtilis (Ehrenberg) Cohn ATCC®7480™ strain NRS 1107deposited with ATCC as Bacillus endoparasiticus by (Benedek) Benedek,Bacillus subtilis (Ehrenberg) Cohn ATCC®8188™ strain ATCC 8450, NRS 773deposited with ATCC as Tyrothrix minimus, Bacillus subtilis (Ehrenberg)Cohn ATCC®8473™ strain NRS 762, Bacillus subtilis (Ehrenberg) CohnATCC®9466™ strain designation: FDA strain PCI 220 [BUCSAV 170, NCIB8159, NRRL B-558, NRS 1088], Bacillus subtilis (Ehrenberg) CohnATCC®9524™ strain 3R9675, NRS 1109, Bacillus subtilis (Ehrenberg) CohnATCC®9799™ strain NCTC 6276, NRS 1125, Bacillus subtilis (Ehrenberg)Cohn ATCC®9858™ strain NRS 237, NCIB 8063, Bacillus subtilis (Ehrenberg)Cohn ATCC®9943™ strain NRS 979, Bacillus subtilis (Ehrenberg) CohnATCC®10774™ strain BU169, NCIB 8872, Bacillus subtilis (Ehrenberg) CohnATCC®10783™ strain NRRL B-543, Bacillus subtilis (Ehrenberg) CohnATCC®11774™ strain NCTC 8236, DSM 2109, Bacillus subtilis (Ehrenberg)Cohn ATCC®11838™ strain AMC 46-A-6 (strain I), NCIB 8850, Bacillussubtilis (Ehrenberg) Cohn ATCC®12100™ strain NCA 1558, ND 957, Bacillussubtilis (Ehrenberg) Cohn ATCC®12432™ strain MB 32, 56R188, ATCC 13597,NCIB 8993, Bacillus subtilis (Ehrenberg) Cohn ATCC®12695™ strain 51-52,Bacillus subtilis (Ehrenberg) Cohn ATCC®12711™ strain PRL B92, Ra,Bacillus subtilis (Ehrenberg) Cohn ATCC®13542™, Bacillus subtilis(Ehrenberg) Cohn ATCC®13933™ strain NRRL B-1471, Bacillus subtilis(Ehrenberg) Cohn ATCC®13952™ strain 1346, Bacillus subtilis (Ehrenberg)Cohn ATCC®14410™ strain 625, NRS 1115 deposited with ATCC as Bacillusborborokoites by ZoBell and Upham, Bacillus subtilis (Ehrenberg) CohnATCC©14415™ strain 569, NRS 1120 deposited with ATCC as Bacillussubmarinus by ZoBell and Upham, Bacillus subtilis (Ehrenberg) CohnATCC®14416™ strain 576, NRS 1121 deposited with ATCC as Bacillusthalassokoites by ZoBell and Upham, Bacillus subtilis (Ehrenberg) CohnATCC®14593™ strain IAM 1145, Bacillus subtilis (Ehrenberg) CohnATCC®14617™ strain A-1625, Bacillus subtilis (Ehrenberg) CohnATCC©14660™ strain C30-1, Bacillus subtilis (Ehrenberg) Cohn ATCC®14662™strain C30-109, Bacillus subtilis (Ehrenberg) Cohn ATCC®14807™ strainMB-155, Bacillus subtilis (Ehrenberg) Cohn ATCC®15040™ strain SX-67,Bacillus subtilis (Ehrenberg) Cohn ATCC®15041™ strain SX-92, Bacillussubtilis (Ehrenberg) Cohn ATCC®15134™ deposited with ATCC as Bacillusuniflagellatus by Mann, Bacillus subtilis (Ehrenberg) Cohn ATCC®15183™strain 309, Bacillus subtilis (Ehrenberg) Cohn ATCC®15244™ strain 3369,Bacillus subtilis (Ehrenberg) Cohn ATCC®15245™ strain 3349, IAM 1-3deposited with ATCC as Bacillus natto by Sawamura, Bacillus subtilis(Ehrenberg) Cohn ATCC®15476™ strain M-4-45, Bacillus subtilis(Ehrenberg) Cohn ATCC®15477™ strain M-24-1 deposited with ATCC asBacillus pumilus by Meyer and Gottheil, Bacillus subtilis (Ehrenberg)Cohn ATCC©15561™ strain K-X-1, A-1, Bacillus subtilis (Ehrenberg) CohnATCC®15563™ strain Marburg, Bacillus subtilis bacteriophage SP8ATCC®15563-B1™ strain SP8 deposited with ATCC as SP8 bacteriophage,Bacillus subtilis (Ehrenberg) Cohn ATCC®15575™ strain SB 19, Bacillussubtilis (Ehrenberg) Cohn ATCC®15811™ strain 5380, Bacillus subtilis(Ehrenberg) Cohn ATCC®15818™ strain RIA 445, Bacillus subtilis(Ehrenberg) Cohn ATCC®15819™ strain RIA 447, Bacillus subtilis(Ehrenberg) Cohn ATCC®15841™, Bacillus subtilis bacteriophage S-aATCC®15841-B1™ strain S-a deposited with ATCC as S-a bacteriophage,Bacillus subtilis (Ehrenberg) Cohn ATCC®19659™ strain PRD 66, IFO 13722,Bacillus subtilis (Ehrenberg) Cohn ATCC®19659-MINI-PACK™ strain PRD 66,IFO 13722, Bacillus subtilis (Ehrenberg) Cohn ATCC®21008™ strain182-H-86 deposited with ATCC as Bacillus pumilus by Meyer and Gottheil,Bacillus subtilis (Ehrenberg) Cohn ATCC®21183™ strain 5221, Bacillussubtilis (Ehrenberg) Cohn ATCC®21228™ strain SC 8548, SO-4, DSM 1970,Bacillus subtilis (Ehrenberg) Cohn ATCC©21331™ strain IFO 35, Bacillussubtilis (Ehrenberg) Cohn ATCC®21332™ strain IAM 1213, Bacillus subtilis(Ehrenberg) Cohn ATCC®21394™ strain 4-3-Ky, DSM 1971 deposited with ATCCas Bacillus subtilis subspecies sakainensis, Bacillus subtilis(Ehrenberg) Cohn ATCC®21555™ strain Y 13, Bacillus subtilis (Ehrenberg)Cohn ATCC®21556™, Bacillus subtilis (Ehrenberg) Cohn ATCC®21742™ strainAHr-5, Bacillus subtilis (Ehrenberg) Cohn ATCC©21770™ strain SP-3deposited with ATCC as Bacillus cereus by Frankland and Frankland,Bacillus subtilis (Ehrenberg) Cohn ATCC®21951™ strain 716, IFO 13322deposited with ATCC as Bacillus pumilus by Meyer and Gottheil, Bacillussubtilis (Ehrenberg) Cohn ATCC®23059™ strain W23, Bacillus subtilis(Ehrenberg) Cohn ATCC©23856™ strain EMG 50, SB19, Bacillus subtilis(Ehrenberg) Cohn ATCC®23857™ strain 168, Bacillus subtilis (Ehrenberg)Cohn ATCC®23857D-5™ strain Designation: Genomic DNA from Bacillussubtilis strain 168 [ATCC® 23857™]Bacillus subtilis (Ehrenberg) CohnATCC®23858™ strain EMG 52, Bacillus subtilis (Ehrenberg) CohnATCC®23859™ strain EMG 53, Bacillus subtilis (Ehrenberg) CohnATCC®25369™ strain 24028 deposited with ATCC as Bacillus pulvifaciens byNakamura, Bacillus subtilis (Ehrenberg) Cohn ATCC®27328™ strain C,Bacillus subtilis (Ehrenberg) Cohn ATCC©27370™ strain 168 M, Bacillussubtilis bacteriophage SPO1 ATCC®27370-B1™ strain SPO1 deposited withATCC as SPO1, Bacillus subtilis (Ehrenberg) Cohn ATCC®27505™ strain K49,HER 1346 deposited with ATCC as Bacillus subtilis subspeciesamyloliquefaciens, Bacillus subtilis (Ehrenberg) Cohn ATCC©27689™ strainSB168 (trp-), Bacillus subtilis (Ehrenberg) Cohn ATCC®29056™ strainSB100, Bacillus subtilis (Ehrenberg) Cohn ATCC®29233™ strain X6,Bacillus subtilis (Ehrenberg) Cohn ATCC®31002™ strain Ahr.AUr-9,FERM-1998, Bacillus subtilis (Ehrenberg) Cohn ATCC®31028™ strain FD 6404deposited with ATCC as Bacillus globigii by Migula, Bacillus subtilis(Ehrenberg) Cohn ATCC®31091™ strain 1054, IFO 13586, Bacillus subtilis(Ehrenberg) Cohn ATCC®31094™ strain 1097, IFO 13621, Bacillus subtilis(Ehrenberg) Cohn ATCC®31098™ strain 1027, IFO 13585 deposited with ATCCas Bacillus pumilus by Meyer and Gottheil, Bacillus subtilis subspeciessubtilis (Ehrenberg) Nakamura et al. ATCC®31578™ strain DSM 6223, RUB331, Bacillus subtilis (Ehrenberg) Cohn ATCC®31954™ strain MO7S-16/11,Bacillus subtilis (Ehrenberg) Cohn ATCC®33234™ strain NCIB 10106,Bacillus subtilis (Ehrenberg) Cohn ATCC®35021™ strain 5230, NRS 6,Bacillus subtilis (Ehrenberg) Cohn ATCC®35854™ strain NRRL B-3411,Bacillus subtilis (Ehrenberg) Cohn ATCC®35946™ strain OSU 75, Bacillussubtilis subspecies subtilis (Ehrenberg) Nakamura et al. ATCC®37014™strain DSM 6224, BD170, pSA2100, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. ATCC®37015™ strain DSM 4514, BD170, NCIB11624, pUB110, Bacillus subtilis subspecies subtilis (Ehrenberg)Nakamura et al. ATCC®37108™ strain DSM 4873, BGSC 1E32, BR151, pPL608,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al.ATCC®37128™ strain DSM 4554, BGSC 1E18, pE194, Bacillus subtilissubspecies subtilis (Ehrenberg) Nakamura et al. ATCC®39090™ strain DSM6198, BGSC 1S53, Bacillus subtilis (Ehrenberg) Cohn ATCC©39320™ strainMB 4488, Bacillus subtilis (Ehrenberg) Cohn ATCC®39374™ strain MB 3575,Bacillus subtilis (Ehrenberg) Cohn ATCC®39706™ strain B1-20, Bacillussubtilis (Ehrenberg) Cohn ATCC®43223™ strain ABM261, Bacillus subtilis(Ehrenberg) Cohn ATCC®49343™ strain IMVS 0101, Bacillus subtilis(Ehrenberg) Cohn ATCC©49760™ deposited with ATCC as Bacillus globigii byMigula, Bacillus subtilis (Ehrenberg) Cohn ATCC®49822™ deposited withATCC as Bacillus globigii by Migula, Bacillus subtilis (Ehrenberg) CohnATCC®55033™ strain SMS274, Bacillus subtilis (Ehrenberg) CohnATCC®55060™ strain MB 4974, Bacillus subtilis (Ehrenberg) CohnATCC®55405™ strain 300, Bacillus subtilis subspecies inaquosorumATCC®55406™ strain DA33 deposited with ATCC as Bacillus licheniformis(Weigmann) Chester, Bacillus subtilis (Ehrenberg) Cohn ATCC®55422™strain SC 15257, Bacillus subtilis (Ehrenberg) Cohn ATCC®55614™ strain1.2, AQ153, Bacillus subtilis (Ehrenberg) Cohn ATCC®55675™ strain BPO1,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strainDSM 402, BRC 111470, NCIB 10106, Bacillus subtilis subspecies spizizeniiNakamura et al. strain DSM 618, Bacillus subtilis subspecies spizizeniiNakamura et al. strain DSM 1087, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 1088, IFO 13169, NBRC 13169, OUT 8353, Bacillus subtilis(Ehrenberg) Cohn strain DSM 1089, IFO 3026, NBRC 3026, OUT 8350,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strainDSM 1090, OUT 8424, Bacillus subtilis subspecies subtilis (Ehrenberg)Nakamura et al. strain DSM 1091, OUT 8425, Bacillus subtilis (Ehrenberg)Cohn strain DSM 1092, IFO 3009, NBRC 3009, OUT 8235, Bacillus subtilissubspecies subtilis (Ehrenberg) Nakamura et al. strain DSM 3256, IAM1213, Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al.strain DSM 3257, IAM 1259, Bacillus subtilis (Ehrenberg) Cohn strain DSM3258, IAM 1260, Bacillus subtilis (Ehrenberg) Cohn strain DSM 4181, NCA72-52, SA 22, Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamuraet al. strain DSM 4393, pC194, SB202, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 4449, natto 3335 UM4, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 4450, natto 3335 UM8, pLS20, pBC16, Bacillus subtilis(Ehrenberg) Cohn strain DSM 4451 Bacillus subtilis (Ehrenberg) Cohnstrain DSM 4515, DB163, pGR71, Bacillus subtilis (Ehrenberg) Cohn strainDSM 4608, BR157, pMW1, Bacillus subtilis subspecies subtilis (Ehrenberg)Nakamura et al. strain DSM 4750, 1E7, BGSC 1E7, pE194-cop6, Bacillussubtilis subspecies subtilis (Ehrenberg) Nakamura et al. strain DSM4751, 1E34, BGSC 1E34, pAM77, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 4871, BD426, BGSC 1E21, pBD8,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strainDSM 4872, BD466, BGSC 1E24, pBD10, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 4874, BGSC 1E38, pMK3, YB886,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strainDSM 5213, BGSC 1A40, BR 151, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 5214, BD 393, BGSC 1A511,Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strainDSM 5545, BGSC 1A459/SU+III, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 5547, Bacillus subtilis(Ehrenberg) Cohn strain DSM 5552, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 5611, NRRL B-360, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 5660, NRRL B-362, Bacillussubtilis subspecies spizizenii Nakamura et al. strain DSM 6395, BGSC2A2, W23 2A2, WB 672, Bacillus subtilis (Ehrenberg) Cohn strain DSM6397, BGSC 1A2, SB 491, Bacillus subtilis subspecies spizizenii Nakamuraet al. strain DSM 6399, BGSC 2A1, SB 623 Bacillus subtilis subspeciesspizizenii Nakamura et al. strain DSM 6405, BGSC 2A3, W23 SR, Bacillussubtilis subspecies subtilis (Ehrenberg) Nakamura et al. strain DSM6887, BGSC 1A309, NP40, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. strain DSM 6889, 1A658, BGSC 1A658, DA 65Bacillus subtilis subspecies spizizenii Nakamura et al. strain DSM 8439,CCM 2268, IAM 12021, Bacillus subtilis (Ehrenberg) Cohn strain DSM13019, SSI MK1, Bacillus subtilis subspecies spizizenii Nakamura et al.strain DSM 15029, NRRL B-23049, Bacillus subtilis subspecies inaquosorumRooney et al. strain DSM 21200, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 21393, Bacillus subtilis subspecies inaquosorum Rooney et al.strain DSM 22148, KCTC 13429, Bacillus subtilis (Ehrenberg) Cohn strainDSM 23521, Bacillus subtilis (Ehrenberg) Cohn strain DSM 23778, Bacillussubtilis (Ehrenberg) Cohn strain DSM 25152, Bacillus subtilis(Ehrenberg) Cohn strain DSM 28592, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30512, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30529,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30533, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30534, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30540, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30541,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30551, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30558, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30562, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30570,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30581, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30597, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30642, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30651,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30652, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30671, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30676, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30677,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30682, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30711, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30723, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30801,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30924, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30925, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 30927, Bacillus subtilis (Ehrenberg) Cohn strain DSM 30928,Bacillus subtilis (Ehrenberg) Cohn strain DSM 30929, Bacillus subtilis(Ehrenberg) Cohn strain DSM 30941, D1, Bacillus subtilis (Ehrenberg)Cohn strain DSM 30942, D-FC1, Bacillus subtilis (Ehrenberg) Cohn strainDSM 31008, Bacillus subtilis (Ehrenberg) Cohn strain DSM 31009, Bacillussubtilis (Ehrenberg) Cohn strain DSM 31010, Bacillus subtilis(Ehrenberg) Cohn strain DSM 31020, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 31021, Bacillus subtilis (Ehrenberg) Cohn strain DSM 31033,Bacillus subtilis (Ehrenberg) Cohn strain DSM 100605, Bacillus subtilis(Ehrenberg) Cohn strain DSM 100612, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 100613, Bacillus subtilis (Ehrenberg) Cohn strain DSM 100614,Bacillus subtilis (Ehrenberg) Cohn strain DSM 103044, Bacillus subtilis(Ehrenberg) Cohn strain DSM 103047, Bacillus subtilis (Ehrenberg) Cohnstrain DSM 103051, Bacillus subtilis (Ehrenberg) Cohn strain DSM 103758,Bacillus subtilis AM0904 (NRRL Deposit Number B-50914), Bacillussubtilis AM0911 (NRRL Deposit Number B-50915), Bacillus subtilis NP122(NRRL Deposit Number B-50910), Bacillus subtilis NP119B (NRRL DepositNumber B-50909), Bacillus subtilis BS18 (NRRL B-50633), Bacillussubtilis BS278 (NRRL 50634), Bacillus subtilis 4-7d (NRRL B-50505),Bacillus subtilis 3-5h (NRRL B-50507), Bacillus subtilis AGTP BS3BP5(NRRL B-50510), Bacillus subtilis BS918 (NRRL B-50508), Bacillussubtilis AGTP BS1013 (NRRL-50509), Bacillus subtilis AGTP 944 (NRRLB-50548), Bacillus subtilis AGTP BS442 (NRRL B-50542), Bacillus subtilisAGTP BS1069 (NRRL B-50544), Bacillus subtilis AGTP BS521 (NRRL B-50545),Bacillus subtilis B27 (NRRL B-50105), Bacillus subtilis 3A-P4(PTA-6506), Bacillus subtilis 22C-P1 (PTA-6508), Bacillus subtilis BL21(NRRL B-50134), Bacillus subtilis strain GB03, Bacillus subtilis strainQST713, Bacillus subtilis DSM 5750 (BioPlus® 2B, Chr. Hansen BioSystems), Bacillus subtilis strain OBT 1224 (Osprey Biotechnic).

Bacillus amyloliquefaciens Strains

Bacillus amyloliquefaciens (Fukumoto) Priest et al. (ATCC® 23350™),Bacillus amyloliquefaciens (Fukumoto) Priest et al. (ATCC® 23842™),Bacillus amyloliquefaciens SB 3296 (PTA-7548), Bacillusamyloliquefaciens (Fukumoto) Priest et al. (ATCC® 23843™), Bacillusamyloliquefaciens SB3297 (PTA-7549), Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® BAA-390™), Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® 23845™) Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® 23844™), Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® 31592™), Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® 53495™), Bacillus amyloliquefaciens(Fukumoto) Priest et al. (ATCC® 49763™) Bacillus amyloliquefaciens: SB3276 (PTA-7541), Bacillus amyloliquefaciens: PMBP-M7 (vial labeled BCRCPMBP-M7) (PTA-5819), Bacillus amyloliquefaciens SB 3284 (PTA-7545),Bacillus amyloliquefaciens SB 3288 (PTA-7546), Bacillusamyloliquefaciens MF215 (SB3446) (PTA-7790), Bacillus amyloliquefaciensSB 3283 (PTA-7544), Bacillus amyloliquefaciens MF 225 (SB 3448)(PTA-7791), Bacillus sp. (ATCC® 70038™, Deposited As Bacillusamyloliquefaciens (Fukumoto) Priest et al.), Bacillus amyloliquefaciensTOA5001 (NITE Patent Microorganisms Depositary Accession NumberBP-01844).

III. Amounts of Bacillus coagulans, Bacillus subtilis, Bacilluslicheniformis and/or Bacillus amyloliquefaciens in Bacilli Combinations

The relative amounts of Bacillus coagulans, Bacillus subtilis, Bacilluslicheniformis and/or Bacillus amyloliquefaciens present in the Bacillicombination are selected to obtain a desired result. For certainembodiments, the Bacilli combination comprises from about 10⁵ to about10¹² CFU/gram, and more typically from about 10⁵ to 10¹⁰ or from 10⁸ to10¹⁰ CFU/gram of each of the Bacillus species in the Bacillicombination. In certain embodiments, the Bacillus combination is addedto feed in an amount sufficient to provide from 5×10⁵ to 8×10⁵ CFU/gramof the feed/Bacillus combination. And in particular embodiments, 0.25pounds/ton of feed is sufficient to provide 5×10⁵ to 8×10⁵ CFU/gram.

In some embodiments, the Bacilli combination may be administered toprovide different CFU ratios of the Bacillus species included therein.In some embodiments, the ratio of Bacillus subtilis:Bacilluslicheniformis in the Bacilli combination may be from 2:1 to 1:2, andtypically is about 1:1, relative to each other. And with respect toother Bacillus species in the Bacilli combination, the total amount ofBacillus subtilis and Bacillus lichemformis (BSBL) relative to the otherBacillus species may be from greater than zero to 99%, such as from 10%to 90%, from 15% to 85%, from 20% to 80%, from 25% to 75%, from 35% to65%, from 45% to 55%, or substantially 50%, based on CFU.

In some embodiments, the ASL combination may comprise, consistessentially of, or consist of, in amounts relative to each other, from25% or less to 75% or more Bacillus amyloliquefaciens (BA) and from 75%or more to 25% or less BSBL. In certain embodiments, the ratio of BA toBSBL in the ASL combination is from 25%:75% BA:BSBL to 75%:25% BA:BSBL,and may be about 50%:50% BA:BSBL.

In some embodiments, the ASLC combination may comprise, consistessentially of, or consist of, in amounts relative to each other, from25% or less to 75% or more in total of Bacillus amyloliquefaciens (BA)and Bacillus coagulans (BC), and from 75% or more to 25% or less BSBL.In certain embodiments, the ratio of BA+BC to BSBL in the ASLcombination is from 25%:75% BA+BC:BSBL to 75%:25% BA+BC:BSBL, and may beabout 50%:50% BA+BC:BSBL. The amounts of BA and BC, relative to eachother may be from greater than zero to 99% BA relative to BC, such asfrom 10% to 90%, from 15% to 85%, from 20% to 80%, from 25% to 75%, from35% to 65%, from 45% to 55%, or substantially 50% BA relative to BC,based on CFU.

For example, the CSL combination may comprise from 3.5×10⁹ to 10×10⁹ CFUBacillus coagulans per gram of the CSL combination, such as from 4.1×10⁹to 7.5×10⁹, from 5×10⁹ to 6.4×10⁹ or from 5×10⁹ to 6×10⁹ CFU Bacilluscoagulans/gram. The CSL combination may comprise from 5×10⁸ to 10×10⁸CFU Bacillus subtilis per gram of the CSL combination, such as from6×10⁸ to 8.7×10⁸, from 6.9×10⁸ to 9×10⁸, or 7.2×10⁸ to 8×10⁸ CFUBacillus subtilis/per gram. And the CSL combination may comprise from5×10⁸ to 10×10⁸ CFU Bacillus licheniformis per gram of the CSLcombination, such as from 6×10⁸ to 8.7×10⁸, from 6.9×10⁸ to 9×10⁸, or7.2×10⁸ to 8×10⁸ CFU Bacillus lichemformis per gram.

In certain embodiments, the CSL combination may be administered toprovide different CFU ratios of the three Bacillus species. For example,in one embodiment, the CSL combination ratio provides from about 6 partsto about 10 parts Bacillus coagulans to 1 part to 2 parts Bacillussubtilis, and from about 1 part to about 2 parts Bacillus licheniformis.The ratio of Bacillus subtilis:Bacillus licheniformis in the CSLcombination may be from 2:1 to 1:2, and typically is about 1:1. Incertain embodiments, the CSL combination comprises about 5×10⁹ Bacilluscoagulans, about 8×10⁸ Bacillus subtilis, and 8×10⁸ Bacilluslicheniformis per gram of the CSL combination.

In other embodiments, the Bacillus combination comprises, consistsessentially of, or consists of, Bacillus subtilis, Bacilluslicheniformis, Bacillus amyloliquefaciens and Bacillus coagulans. Therelative amounts of the Bacillus species in such embodiments are from25% to 50% Bacillus subtilis, from 30% to 65% Bacillus lichemformis,from 5% to 30% Bacillus amyloliquefaciens and from greater than zero to15% Bacillus coagulans, in amounts relative to each other, such as from30% to 45% Bacillus subtilis, from 40% to 60% Bacillus licheniformis,from 10% to 25% Bacillus amyloliquefaciens and from 1% to 12% Bacilluscoagulans. In certain embodiments, the Bacillus combination comprises,consists essentially of, or consists of, from 30% to 40% Bacillussubtilis, from 40% to 50% Bacillus lichemformis, from 10% to 20%Bacillus amyloliquefaciens and from 2% to 10% Bacillus coagulans inamounts relative to each other. In some embodiments, the amount ofBacillus licheniformis is greater than the amount of Bacillus subtilisin the Bacillus combination. In certain embodiments, such a compositioncan be a commercially available product, such as the composition sold asProvia Prime™ by Phibro Animal Health Corporation.

IV. Additional Component(s)

The Bacilli combination also can be administered in combination with oneor more additional components or compositions. An additional componentor composition may be any component or composition that can beadministered to a subject, particularly an animal, such as an avian,including poultry, in combination with the Bacilli species in theBacilli combination, such as Bacillus coagulans, Bacillus subtilis andBacillus licheniformis, or Bacillus amyloliquefaciens, Bacillus subtilisand Bacillus licheniformis and optionally Bacillus coagulans. Certaindisclosed Bacilli combinations are particularly formulated foradministration to poultry, and therefore can comprise the Bacillicombination in combination with any other component or composition nowknown or hereafter developed for administration to poultry. Exemplaryadditional components include a carrier, a vitamin, a copper salt, afeed supplement, an additional DFM, a feed, such as a poultry feed, or acombination thereof. The additional component(s) will comprise from 1 wt% to 99 wt % and the Bacilli combination will comprise from 99 wt % to 1wt % of the total weight of the combination. Preferably the additionalcomponent(s) will comprise from 10 wt % to 90 wt % and the Bacillicombination will comprise from 90 wt % to 10 wt % of the total weight ofthe combination. Yet even more preferably, the additional component(s)will comprise 20 wt % to 80 wt % and the Bacilli combination willcomprise from 80 wt % to 20 wt % of the total weight of the combination.The Bacilli combination may be administered with the other component(s),optionally in a mixture with the other component(s), such as poultryfeed and/or a feed supplement, in an amount sufficient to provide thedesired amounts of the respective Bacillus species in the particularcombination. For example for the CSL combination, the amountadministered may be sufficient to provide from 0.5×10⁵ to 2×10⁵ CFUBacillus coagulans per gram of the mixture, from 1.2×10⁵ to 4×10⁵ CFUBacillus subtilis per gram of the mixture, and/or from 1.2×10⁵ to 4×10⁵CFU Bacillus licheniformis per gram of the mixture. In some embodiments,the amount of Bacillus coagulans in the mixture is from 0.6×10⁵ to1.5×10⁵ CFU, or from 0.9×10⁵ to 1.2×10⁵ CFU per gram of the mixture.Independently, the amount of each of the Bacillus subtilis and Bacilluslichemformis in the mixture may be from 1.5×10⁵ to 3×10⁵ or from 2.3×10⁵to 3×10⁵ CFU per gram of the mixture. In certain embodiments, the CSLcombination is administered with one or more additional components, suchas feed and/or feed supplement, in an amount sufficient to provide0.62×10⁵, 0.93×10⁵ or 1.2×10⁵ CFU Bacillus coagulans, and independently1.5×10⁵, 2.3×10⁵ or 3×10⁵ CFU of each of the Bacillus subtilis andBacillus lichemformis, per gram of the mixture. Exemplary additionalcomponents and/or compositions of a combination comprising the Bacillicombination are discussed in more detail below.

A. Carrier

In some embodiments, the Bacilli combination may be mixed with and/ordispersed in a carrier to form a dispersed composition. The carrier(s)may be selected to provide a non-biological benefit to the composition,compared to a Bacilli combination without a carrier, such as, but notlimited to, achieving or improving a readily flowable state, and/orimproving stability during storage and/or transport. Suitable carriersthat may be used in combination with a Bacilli combination include, butare not limited to, plant material, such as beet pulp, ground corn, cornsyrup solids, plant fiber, rice hulls, soluble plant fiber, wheatmiddlings, microcrystalline cellulose; carbonates, such as metalcarbonates, such as calcium carbonate, potassium carbonate; sulfates,such as metal sulfates, such as potassium sulfate, sodium sulfate;lactates, including metal lactates, such as calcium lactate; oxides,including metal oxides, such as calcium oxide; propionates, includingmetal propionates, such as calcium propionate; stearates, includingmetal stearates, such as calcium stearate; phosphates, such as dicalciumphosphate dehydrate, monocalcium phosphate, sodium tripolyphosphate, ortetra sodium pyrophosphate; minerals, such as dolomite, silicon dioxide,silica, limestone, or vermiculite; clays, such as bentonite,montmorillonite, kaolin; sugars, such as glucose, sucrose, dextrose,fructose, or a combination thereof; maltodextrin; salt, such as sodiumchloride; carrageenan; cellulose; guar gum; polyols; sodium aluminosilicate; urea; animal protein products; forage products; grainproducts; plant protein products; processed grain products; roughageproducts; molasses products; or combinations thereof. In certainembodiments, the carrier is calcium carbonate.

Animal protein products may include, but are not limited to, blood meal;animal by-product meal; buttermilk, including condensed buttermilk anddried buttermilk; casein; dried hydrolyzed casein; cheese rind; crabmeal; fish products, including fish by-products, fish liver andglandular meal, fish meal, fish protein concentrates, fish residue meal,and dried and/or condensed fish solubles; fleshings hydrolysate;hydrolyzed hair; hydrolyzed leather meal; hydrolyzed poultry by-productaggregate; hydrolyzed poultry feathers; leather hydrolysate; meat andbone meal; meat and bone meal tankage; meat meal; meat meal tankage;dried meat solubles; dried lactalbumin; dried feed grade milk; driedmilk protein; poultry by-products and/or by-products meal; poultryhatchery by-product; shrimp meal; skimmed milk, including condensed,condensed cultured, dried, or dried cultured skimmed milk; whey,including condensed, condensed cultured, condensed hydrolyzed, dried, ordried hydrolyzed whey; condensed and/or dried whey product; condensedand/or dried whey solubles; or a combination thereof.

Forage products may include, but are not limited to, alfalfa products,such as dehydrated meal, optionally in pellet form, ground hay, orsuncured meal, optionally in pellet form; coastal bermudagrass hay;dehydrated corn plant; dehydrated silage; flax plant product; groundgrass; lespedeza meal and/or stem meal; ground soybean hay; orcombinations thereof.

Grain products may include, but are not limited to, barley, corn, grainsorghum, mixed feed oats, oats, triticale, wheat, ground brown rice,ground or ground paddy rough rice, broken or chipped rice, brewers rice,rye, or a combination thereof. The grain products may be in any suitableform, such as whole, ground, cracked, hulls, bran, screen cracked,flaked, kibbled, toasted, and/or heat processed.

Plant protein products may include, but are not limited to, dried beans;canola meal; coconut meal; cottonseed, such as flakes, cake, meal, lowgossypol meal, and/or whole pressed cottonseed; guar meal; dried kelp;linseed meal; peanut meal; peas; potato protein; dried seaweed meal;safflower meal; soy protein concentrate; soybean feed; ground soybeans;soybean meal, optionally kibbled; heat processed soybeans; ground,extruded whole soybeans; soy flour; soy grits; sunflower meal,optionally dehulled; or a combination thereof.

The processed grain by-products may be aspirated grain fractions;brewers dried grains; buckwheat middlings; condensed distillerssolubles; condensed fermented corn extracts; corn bran; corn flour; corngerm meal; corn gluten feed and/or meal; corn grits; distillers driedgrains, optionally with solubles; distillers dried solubles, flour,grain sorghum germ cake, meal, grits, and/or mill feed; meal hominyfeed; malt sprouts; oat groats; feeding oat meal; pearl barleyby-product; peanut skins; rice bran; rice polishings; rye middlings;gelatinized or partially aspirated sorghum grain flour; wheat bran,flour, shorts, germ meal, defatted germ meal, middlings, mill run and/orred dog; or a combination thereof.

Roughage products may include, but are not limited to, almond hulls;dried apple pectin pulp; dried apple pomace; bagasse; barley hulls;barley mill by-product; dried, plain beet pulp; buckwheat hulls; driedcitrus meal; dried citrus pulp; citrus seed meal; corn cob fractions;cottonseed hulls; flax straw by-product; ground corn cob; psyllium seedhusk; malt hulls; clipped oat by-product; oat hulls; oat millby-product; peanut hulls; rice hulls; rice mill by-product; rye millrun; soybean hulls, mill feed, and/or mill run; sunflower hulls; groundstraw; dried tomato pomace; or a combination thereof.

Molasses products may be beet molasses; dried beet molasses product;dried beet pulp molasses; cane molasses; citrus molasses; molasses yeastcondensed solubles; concentrated separator by-product; condensedmolasses fermentation solubles; starch molasses; molasses distillerscondensed solubles; molasses distillers dried solubles; or a combinationthereof.

B. Copper Species

The disclosed combination may be mixed with a copper species such as acopper species that provides a copper ion. The copper species may be acopper salt. Exemplary copper species that may be combined with theBacilli combination include, but are not limited to, copper chloride,copper bromide, copper iodide, copper sulfate, copper sulfite, copperbisulfite, copper thiosulfate, copper phosphate, monobasic copperphosphate, dibasic copper phosphate, copper hypophosphite, copperdihydrogen pyrophosphate, copper tetraborate, copper borate, coppercarbonate, copper bicarbonate, copper metasilicate, copper citrate,copper malate, copper methionate, copper succinate, copper lactate,copper formate, copper acetate, copper butyrate, copper propionate,copper benzoate, copper tartrate, copper ascorbate, copper gluconate, ora combination thereof, preferably copper sulfate, copper acetate, coppercitrate, copper methionate, or a combination thereof. A copper species,such as a copper salt, may be provided separately, or individually, orit may be provided as part of a composition, such as a feed or a feedsupplement. Certain disclosed embodiments comprise, consist essentiallyof, or consist of Bacillus coagulans, Bacillus subtilis, Bacilluslicheniformis and a copper species. Other particular embodiments,comprise, consist essentially of, or consist of Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus licheniformis and acopper species. And other particular embodiments, comprise, consistessentially of, or consist of Bacillus amyloliquefaciens, Bacillussubtilis, Bacillus licheniformis and Bacillus coagulans and a copperspecies. In any embodiments, the copper species may be a copper salt,such as a salt that can provide a copper ion, for example, coppersulfate.

C. Vitamin(s)

Exemplary vitamins include, but are not limited to, one or more ofVitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3(niacin or niacinamide), Vitamin B5 (pantothenic acid), Vitamin B6(pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride),Vitamin B7 (biotin), Vitamin B9 (including folic acid), Vitamin B12(various cobalamins; commonly cyanocobalamin in vitamin supplements),Vitamin C (ascorbic acid or a salt thereof, such as sodium ascorbate orcalcium sorbate), Vitamin D (vitamin D₁, vitamin D₂, vitamin D₃, vitaminD₄, vitamin D₅, 25-hydroxy vitamin D₃, 25-dihydroxy vitamin D₃, orcombinations thereof), Vitamin E, Vitamin K (K1 and K2 (i.e. MK-4,MK-7)), and biotin, and derivatives, salts and/or analogs thereof.

D. Feed

The feed may be any feed suitable for administration to an animal. TheBacilli combination may be administered in combination with the feed,such as by forming a mixture of the Bacilli combination and the feed, orby administering the Bacilli combination and the feed sequentially, inany order. In certain disclosed embodiments the animal is a poultry, andthe Bacilli combination is used in combination with, and may be admixedwith, a poultry feed, such as a poultry basal diet. The feed maycomprise corn, alfalfa, peas, soybean meal, soybean oil, wheat, oats,sorghum, barley, rye, rice hulls, canola, corn oil, limestone, salt (forexample, sodium chloride), distillers dried grains with solubles (DDGS),dicalcium phosphate, sodium sesquicarbonate, methionine source, lysinesource, L-threonine, mineral oil, biotin, folic acid, kelp, menadionedimethylpyrimidinol bisulfite, calcium aluminosilicate, or anycombination thereof. The feed may also comprise one or more additionalcomponents. Additional components may be used for any desired purpose,such as a substantially biologically inert material added, for example,as a filler, or to provide a desired beneficial effect. For example, thefeed may include a carbonate (including a metal carbonate such ascalcium carbonate); a trace mineral, such as, but not limited to,chloride, fluoride, iodide, chromium, copper, zinc, iron, magnesium,manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium,or a combination thereof; a bulking agent; a carrier; a colorant; ataste enhancer; a preservative; one or more vitamins; or a combinationthereof. The preservative may be benzoic acid or a salt thereof, e.g.sodium benzoate; lactic acid or a salt thereof, e.g. sodium lactate,potassium lactate or calcium lactate; propionic acid or a salt thereof,e.g. sodium propionate; ascorbic acid or a salt thereof, e.g. sodiumascorbate; gallic acid or a salt thereof e.g. sodium gallate; sulfurdioxide and/or sulfites; nitrites; nitrates; choline, or a salt thereof,such as an anion salt of choline, e.g. choline halide, such as chloride,bromide, iodide, fluoride, or choline hydroxide; or any combinationthereof. The one or more vitamins may include vitamin A; vitamin B₁,such as thiamine mononitrate; vitamin B₂, such asriboflavin-5-phosphate; vitamin B₃, such as niacin or niacinamide;vitamin B₅, such as pantothenic acid or d-calcium pantothenate; vitaminB₆, such as pyridoxine or pyridoxine hydrochloride; vitamin B₁₂; vitaminC, such as ascorbic acid, sodium ascorbate, or calcium sorbate; vitaminD; vitamin E; vitamin K, or a combination thereof. Vitamin D maycomprise vitamin D₁, vitamin D₂, vitamin D₃, vitamin D₄, vitamin D₅,25-hydroxy vitamin D₃, 25-dihydroxy vitamin D₃, or combinations thereof.

The feed, such as a poultry feed, may also include fats and/or oils,such as tallow, optionally derived from the rendering of beef offal;lard, optionally derived from the rendering of pork offal; poultry fat,optionally derived from poultry offal; feed grade animal fat, optionallyderived from a mixture of rendered beef, pork, and/or poultry rawmaterial; yellow grease, optionally derived from reprocessed restaurantgrease and/or cooking oil; and/or blended animal-vegetable fat, whichmay include blends of different types and/or amounts of animal fats andvegetable oils from restaurant grease. Additionally, or alternatively,the feed may include protein sources, such as canola, fish meal, fieldpeas, meat and bone meal, soybeans, and/or cereal by-products.

E. Feed Supplements

The Bacilli combination may be used in combination with one or more feedsupplements. In some embodiments, the Bacilli combination is mixed withthe feed supplement to form a mixture or composition comprising theBacilli combination and the feed supplement(s). In other embodiments,the Bacilli combination is administered in combination with a feedsupplement.

1. Yucca and/or Quillaja, or Extracts Thereof

Additionally, or alternatively, a disclosed Bacilli combination can beadministered in combination with yucca and/or quillaja plant material,or extracts thereof. Examples of yucca include, but are not limited to,Yucca aloifolia, Yucca angustissima, Yucca arkansana, Yucca baccata,Yucca baileyi, Yucca brevifolia, Yucca campestris, Yucca capensis, Yuccacarnerosana, Yucca cernua, Yucca coahuilensis, Yucca constricta, Yuccadecipiens, Yucca declinata, Yucca desmetiana, Yucca elata, Yuccaendlichiana, Yucca faxoniana, Yucca filamentosa, Yucca filifera, Yuccaflaccida, Yucca gigantean, Yucca glauca, Yucca gloriosa, Yuccagrandiflora, Yucca harrimaniae, Yucca intermedia, Yucca jaliscensis,Yucca lacandonica, Yucca linearifolia, Yucca luminosa, Yucca madrensis,Yucca mixtecana, Yucca necopina, Yucca neomexicana, Yucca pallida, Yuccapericulosa, Yucca potosina, Yucca queretaroensis, Yucca reverchonii,Yucca rostrata, Yucca rupicola, Yucca schidigera, Yucca schottii, Yuccasterilis, Yucca tenuistyla, Yucca thompsoniana, Yucca treculeana, Yuccautahensis, Yucca valida or combinations thereof. In certain embodiments,the Yucca is or comprises Yucca schidigera.

Examples of quillaja include, but are not limited to, Quillajabrasiliensis, Quillaja lanceolata, Quillaja lancifolia, Quillajamolinae, Quillaja petiolaris, Quillaja poeppigii, Quillaja saponaria,Quillaja sellowiana, Quillaja smegmadermos or combinations thereof. Incertain embodiments, the quillaja is or comprises Quillaja saponaria.

A person of ordinary skill in the art will appreciate that, as usedherein, a plant name may refer to the plant as a whole, or to any partof the plant, such as the roots, stem or trunk, bark, leaves, flower,flower stems, seeds, or a combination thereof. These plant parts may beused fresh, or dried, and may be whole, pulverized, or comminuted. Theplant name may also refer to extracts from any part or parts of theplant, such as chemical extracts, or extracts obtained by pressing, orany other methods of concentrating or extracting oils or other extractsknown to those in the art or that are hereafter discovered. Plantextracts may include compounds that are saponins, triterpenoids,polyphenols, antioxidants or resveratrol, or combinations thereof.

The combination may comprise a composition comprising yucca and/orquillaja that may also include carriers and binding agents suitable toformulate the yucca and/or quillaja for administration to an animal. Incertain embodiments, such a composition can be a commercially availableproduct, such as a composition comprising Yucca schidigera and Quillajasaponaria, sold under the trademark NUTRAFITO PLUS by Desert KingInternational and/or MAGNI-PHI© by Phibro Animal Health Corporation.Such compositions may comprise from 99% or more Quillaja saponaria and1% or less Yucca schidigera to 75% Quillaja saponaria and 25% Yuccaschidigera, such as from 95% Quillaja saponaria and 5% Yucca schidigerato 80% Quillaja saponaria and 20% Yucca schidigera, and in certainembodiments, 85% Quillaja saponaria and 15% Yucca schidigera, or 90%Quillaja saponaria and 10% Yucca schidigera.

In some embodiments, a combination and/or composition comprises,consists essentially of, or consists of, yucca, quillaja, Bacilluscoagulans, Bacillus subtilis, Bacillus licheniformis and Bacillusamyloliquefaciens, such as Yucca schidigera, Quillaja saponaria,Bacillus coagulans, Bacillus subtilis, Bacillus licheniformis andBacillus amyloliquefaciens. The combination and/or composition maycomprise, consist essentially of, or consist of, from 100 to 500 ppm ormore yucca and quillaja, and from 0.1 to 1 pounds of Bacillus coagulans,Bacillus subtilis, Bacillus licheniformis and Bacillusamyloliquefaciens, per ton of a feed, such as from 200 to 500 ppm yuccaand quillaja, and from 0.1 to 0.5 pounds of Bacillus coagulans, Bacillussubtilis, Bacillus licheniformis and Bacillus amyloliquefaciens, and incertain embodiments, from 200 to 300 ppm Yucca schidigera and Quillajasaponaria, and from 0.2 to 0.3 pounds of Bacillus coagulans, Bacillussubtilis, Bacillus licheniformis and Bacillus amyloliquefaciens.

2. Silica, Mineral Clay, Glucan and Mannans

Additionally, or ahernatively, a Bacilli combination can be administeredin combination with a feed supplement comprising silica, mineral clay,glucan and mannans. The feed supplement may further comprise anendoglucanohydrolase, either endogenously or as an affirmatively addedingredient. As used herein, weight % for endoglucanohydrolase is basedon a 70,000 unit/gram endoglucanohydrolase product. Theendoglucanohydrolase may be β-1,3 (4)-endoglucanohydrolase.

In any embodiments disclosed herein, the feed supplement may comprise,consist essentially of, or consist of, glucan (e.g., β-1,3 (4)glucan),silica, mineral clay and mannans. In some embodiments, the feedsupplement comprises, consists essentially of, or consists of, glucan(e.g., β-1,3 (4)glucan), silica, mineral clay, mannans andendoglucanohydrolase. In any embodiments disclosed herein, the glucanand mannans may be provided, at least in part, by yeast cell wall or anextract thereof. Thus, in some embodiments, the feed supplement maycomprise, consist essentially of, or consist of, silica, mineral clayand yeast cell wall or an extract thereof, or the feed supplement maycomprise, consist essentially of, or consist of, silica, mineral clay,yeast cell wall or an extract thereof, and endoglucanohydrolase.Similarly, endoglucanohydrolase may, in certain disclosed embodiments,be provided by yeast cell wall or a yeast cell wall extract.

Suitable sources of silica include, but are not limited to, sand,diatomaceous earth, and synthetic silica. In one embodiment, quartz maybe used. In certain embodiments, the mannans comprise glucomannan.

The components of the feed supplement are prepared by methods commonlyknown in the art and can be obtained from commercial sources. β-1,3(4)-endoglucanohydrolase may be produced from submerged fermentation ofa strain of Trichoderma longibrachiatum. Diatomaceous earth is availableas a commercially-available product with from 70% to 95% silica (SiO₂)and with its remaining components not assayed but primarily ash(minerals) as defined by the Association of Analytical Chemists (AOAC,2002). The mineral clays (e.g., aluminosilicates) used in this feedsupplement may be any of a variety of commercially-available claysincluding, but not limited to, montmorillonite clay, bentonite andzeolite. Glucan, mannans, and/or endoglucanohydrolase can be obtainedfrom plant cell walls, yeast or yeast cell wall or an extract thereof(e.g., Saccharomyces cerevisiae, Candida utilis), certain fungi (e.g.,mushrooms), algae, and bacteria. In certain embodiments, yeast can beadministered affirmatively to provide glucan, mannans andendoglucanohydrolase endogenously.

In one embodiment, the feed supplement comprises, consists essentiallyof, or consists of, 1-40 wt % silica, 0.5-25 wt % glucan and mannans,and 40-92 wt % mineral clay, in amounts relative to each other. Inanother embodiment, the feed supplement comprises, consists essentiallyof, or consists of, 5-40 wt % silica, 0.5-15 wt % glucan and mannans,and 40-80 wt % mineral clay, in amounts relative to each other. Inanother embodiment, the feed supplement comprises, consists essentiallyof, or consists of, 20-40 wt % silica, 0.5-10 wt % glucan and mannans,and 50-70 wt % mineral clay, in amounts relative to each other. Inanother embodiment, the feed supplement comprises, consists essentiallyof, or consists of, 15-40 wt % silica, greater than zero to 15 wt %glucans, greater than zero to 10 wt % mannans, and 50-81 wt % mineralclay, in amounts relative to each other. In another embodiment, the feedsupplement comprises, consists essentially of, or consists of, 15-40 wt% silica, 0.5-5.0 wt % glucans, 0.5-8.0 wt % mannans, and 50-81 wt %mineral clay, in amounts relative to each other. In another embodiment,the feed supplement comprises, consists essentially of, or consists of,20-30 wt % silica, 0.5-3.5 wt % glucans, 0.5-6.0 wt % mannans, and 60-70wt % mineral clay, in amounts relative to each other.

In some embodiments, β-glucans and mannans are obtained from yeast oryeast cell wall or an extract thereof. The feed supplement may comprise,consist essentially of, or consist of, 1-40 wt % silica, 1-30 wt % yeastcell wall or an extract thereof, and 40-92 wt % mineral clay, in amountsrelative to each other. In one embodiment, the feed supplementcomprises, consists essentially of, or consists of, 10-40 wt % silica,5-20 wt % yeast cell wall or an extract thereof, and 40-80 wt % mineralclay, in amounts relative to each other. In another embodiment, the feedsupplement comprises, consists essentially of, or consists of, 15-30 wt% silica, 5-15 wt % yeast cell wall or an extract thereof, and 50-70 wt% mineral clay, in amounts relative to each other.

In any of the above embodiments, the feed supplement may furthercomprise an endoglucanohydrolase, such as β-1,3(4)-endoglucanohydrolase. The feed supplement may include from 0.025 wt% endoglucanohydrolase to 5 wt % endoglucanohydrolase or more, such asfrom 0.05 wt % to 3 wt % β-1,3 (4)-endoglucanohydrolase, relative to theamounts of silica, mineral clay, glucan, mannans, and/or yeast, yeastcell wall, or yeast cell wall extract present in the feed supplement. Inone embodiment, the feed supplement comprises, consists essentially of,or consists of, 0.1-3 wt % β-1,3 (4)-endoglucanohydrolase, 20-40 wt %silica, 0.5-20 wt % glucan and mannans, and 50-70 wt % mineral clay, inamounts relative to each other. In another embodiment, the feedsupplement comprises, consists essentially of, or consists of, 0.1-3 wt%, β-1,3 (4)-endoglucanohydrolase, 20-40 wt % silica, 0.5-10 wt % glucanand mannans, and 50-70 wt % mineral clay, in amounts relative to eachother. Alternatively, the feed supplement may comprise, consistessentially of, or consist of, 0.1-3 wt % β-1,3(4)-endoglucanohydrolase, 1-40 wt % silica, 5-30 wt % yeast cell wall oran extract thereof, and 40-92 wt % mineral clay, in amounts relative toeach other. In one embodiment, the feed supplement comprises, consistsessentially of, or consists of, 0.1-3 wt % β-1,3(4)-endoglucanohydrolase, 10-40 wt % silica, 5-20 wt % yeast cell wallor an extract thereof, and 40-80 wt % mineral clay, in amounts relativeto each other. In another embodiment, the feed supplement comprises,consists essentially of, or consists of, 0.1-3 wt % β-1,3(4)-endoglucanohydrolase, 15-30 wt % silica, 5-15 wt % yeast cell wallor an extract thereof, and 50-70 wt % mineral clay, in amounts relativeto each other.

In any of the above embodiments, the silica may be provided bydiatomaceous earth. In any of the above embodiments, the glucans may beβ-glucans. In some embodiments, the β-glucans can be obtained fromyeast, or other materials, such as fungi, algae, bacteria, or the like.In any of the above embodiments, the mannans may comprise glucomannan.

The glucan and mannans (or yeast or yeast cell wall or an extractthereof) can be prepared by a method known to a person of ordinary skillin the art and as further disclosed by the patent documents incorporatedherein by reference. Yeast cell wall or an extract thereof may have afeed supplement comprising 0-15% moisture and 85-100% dry matter. Thedry matter may comprise 10-65% protein, 0-25% fats, 0-3% phosphorus,5-30% β-glucan, 5-35% mannans, and 0-15% ash. In an independentembodiment, a commercial source of β-1,3 (4) glucan and glucomannanderived from primary inactivated yeast (Saccharomyces cerevisiae) withthe following chemical feed supplement can be used: moisture 2-5%;proteins 40-50%; fats 3-8%; phosphorus 0-2%; mannans 10-16%; β-1,3-(4)glucan 10-20%; and ash 2-12%.

In another independent embodiment, the yeast cell wall or an extractthereof comprises moisture 1-7% and dry matter 93-99%, and the drymatter may comprise proteins 18-28%, fats 10-17%, phosphorus 0-2%,mannans 20-30%, β-1,3-(4) glucan 18-28%, and ash 2-5%.

In an independent embodiment of the feed supplement, silica, glucan andmannans, and mineral clay are combined at 1-40%, 0.5-25% and 40-92% byweight, respectively. In an independent embodiment of the feedsupplement and/or combination, β-1,3 (4)-endoglucanohydrolase,diatomaceous earth, yeast cell wall or an extract thereof, and mineralclay are combined at 0.05-3%, 1-40%, 1-20% and 40-92% by weight,respectively. In an independent feed supplement and/or combination,β-1,3 (4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall oran extract thereof, and mineral clay are combined at 0.1-3%, 5-40%,2-15% and 40-80% by weight, respectively. In another independentembodiment of the feed supplement and/or combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or anextract thereof, and mineral clay are combined at 0.1-3%, 30-40%, 4-15%and 50-65% by weight, respectively.

The feed supplement may further comprise one or more additionalcomponents. Additional components may be used for any desired purpose,such as a substantially biologically inert material added, for example,as a filler, or to provide a desired beneficial effect. For example, thefeed supplement may include a carbonate (including a metal carbonatesuch as calcium carbonate); a trace mineral, such as, but not limitedto, chloride, fluoride, iodide, chromium, copper, zinc, iron, magnesium,manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium,or a combination thereof; a bulking agent; a micro tracer, such as ironparticles coated with a dye; yeast; allicin; alliin; allinase; algae; apolyphenol or plant material comprising polyphenol; a carrier; acolorant; a taste enhancer; a preservative; an oil; a vitamin; a sorbicacid or a salt thereof; or a combination thereof. The yeast may be yeastculture, active yeast, a live yeast, a dead yeast, yeast extract, activedried yeast, brewers dried yeast, culture yeast, dried yeast, primarydried yeast, torula dried yeast, candida dried yeast, or a combinationthereof. The preservative may be benzoic acid or a salt thereof, e.g.sodium benzoate; lactic acid or a salt thereof, e.g. sodium lactate,potassium lactate or calcium lactate; propionic acid or a salt thereof,e.g. sodium propionate; ascorbic acid or a salt thereof, e.g. sodiumascorbate; gallic acid or a salt thereof e.g. sodium gallate; sulfurdioxide and/or sulfites; nitrites; nitrates; choline, or a salt thereof,such as an anion salt of choline, e.g. choline halide, such as chloride,bromide, iodide, fluoride, or choline hydroxide; or any combinationthereof. The oil may be mineral oil, corn oil, soybean oil, or acombination thereof. The sorbic acid or salt thereof may be potassiumsorbate, sodium sorbate, ammonium sorbate, or a combination thereof. Thevitamin may be vitamin A, vitamin B1, vitamin B₂, vitamin B₃, vitaminB₅, vitamin B₆, vitamin B₁₂, vitamin C, vitamin D, vitamin E, vitamin K,or a combination thereof.

Allicin (diallyl thiosulfate; 2-Propene-1-sulfinothioic acidS-2-propenyl ester) is a compound found in garlic, such as raw garlic.Allicin is typically produced from alliin((2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid) in damagedgarlic cells by the action of the enzyme alliinase. Allicin, alliin,and/or alliinase may be provided as whole garlic cloves or bulbs;crushed, mashed, or chopped garlic; a garlic extract; and/or as asynthesized or isolated compound.

The polyphenol may be provided by a plant extract from apolyphenol-containing plant material. The plant material also mayinclude non-polyphenol compounds, including polyphenol degradationproducts, such as gallic acid and trans-caftaric acid. Degradation canoccur, for example, through oxidative and/or biological processes. Boththe polyphenols and the non-polyphenol compounds may have biologicalactivity. The plant extract may be prepared from a single plant materialor from a combination of plant materials. Suitable plant materials fromwhich a plant extract can be obtained include, but are not limited to,apples, blackberries, black chokeberries, black currants, blackelderberries, blueberries, cherries, cranberries, grapes, green tea,hops, onions, quillaja, plums, pomegranates, raspberries, strawberries,and yucca.

In some embodiments, the plant extract is prepared from a pressed plantmaterial, such as grape pomace, a dried plant material, such as tea, ora combination thereof. Pomace may be obtained substantially immediatelypost-pressing or as an ensiled product, i.e., pomace collected andstored for up to several months post-pressing. Suitable plants have aplurality of polyphenols and/or other non-polyphenolic compoundsincluding, but not limited to, non-polyphenolic organic acids (such asgallic acid and/or trans-caftaric acid), flavanols, gallate esters,flavanodiols, phloroglucinol, pyrogallol, and catechol. In someembodiments, the plant extract is prepared from Pinot noir pomace, Pinotgris pomace, or green tea.

In some embodiments, pressed or dried plant material is ground to a finepowder prior to, or during, extraction. Pressed plant materials may befrozen to facilitate grinding. Polyphenols and other non-polyphenoliccompounds may be extracted for administration. For example, polyphenolsand other non-polyphenolic compounds may be extracted from the powderusing a solution comprising a polar solvent, such as water, an alcohol,an ester, or a combination thereof. In some embodiments, the solutioncomprises a water-miscible alcohol, ester, or combination thereof, suchas a lower alkyl alcohol, lower alkyl ester, or a combination thereof.In some embodiments, the solution is water or an aqueous solutioncomprising 25-99% solvent, such as 25-95% solvent, 30-80% solvent, or50-75% solvent, and water. In certain embodiments, the solution is anaqueous solution comprising methanol, ethanol, isopropanol, ethylacetate, or a combination thereof. The solution may be acidified byaddition of an acid. The acid may prevent or minimize oxidativedegradation of biologically-active polyphenols and othernon-polyphenolic compounds in the extract. The acid may be any suitableacid, such as a mineral acid (e.g., hydrochloric acid), or an organicacid such as citric acid or acetic acid. In some embodiments, thesolution comprises from 0.01% to 1% acid, such as 0.02-0.5%,0.025-0.25%, or 0.05-0.15%. In some examples, the solution includes 0.1%hydrochloric acid.

Extraction may be performed at a temperature ranging from 0-100° C. Insome embodiments, extraction is performed at a temperature ranging from20-70° C., or at ambient temperature. Extraction may be performed for aduration ranging from several minutes to several days. To increaseextraction efficiency, the plant material and solution may be mixed oragitated during extraction, such as by grinding the plant materialduring extraction, stirring the mixture, shaking the mixture, orhomogenizing the mixture. In some embodiments, the extraction may berepeated one or more times with fresh solution to increase recovery ofpolyphenols and other non-polyphenolic compounds from the plantmaterial. The liquid phases from each extraction cycle are then combinedfor further processing.

The liquid phase can be recovered, and the residual solids, or pulp, arediscarded. Recovering the liquid phase may comprise decanting the liquidfrom the remaining solids and/or filtering the liquid phase to removeresidual solids. The solvent (alcohol, ester, or combination thereof)can be removed from the liquid solution by any suitable means, such asevaporation (e.g., roto-evaporation), to produce an aqueous extractcontaining the biologically-active components in a mildly acidicsolution.

In certain embodiments where the plant material includes a significantamount of oils, or lipids, an initial extraction of nonpolar componentsmay be performed before extracting the polyphenols and other polar,non-polyphenolic compounds. Nonpolar components may be extracted byhomogenizing the plant material in a nonpolar solvent, e.g., hexanes,heptanes, or a combination thereof. The solvent layer including theextracted nonpolar components is separated from the plant material anddiscarded.

The aqueous plant extract may be further purified by suitable means,e.g., extraction, chromatographic methods, distillation, etc., to removenon-polyphenolic compounds and/or to increase the concentration ofpolyphenols relative to other compounds in the extract.

The aqueous plant extract may be dried, for example by freeze-drying orother low-temperature drying methods, and ground to a powder to providea dried plant extract. In some embodiments, the dried plant extractcomprises 0.01 wt % to 25 wt % total polyphenols, such as 0.01 wt % to10 wt %, 0.01 wt % to 5 wt %, 0.01 wt % to 2.5 wt %, 0.01 wt % to 1 wt%, 0.01 wt % to 0.5 wt %, 0.02 to 0.25 wt %, or 0.03-0.1 wt % totalpolyphenols. In certain embodiments, the dried plant extract furthercomprises non-polyphenolic compounds. For example, the dried plantextract may comprise 0.01-1 mg/g gallic acid, such as 0.05-0.5 mg/g or0.09-0.25 mg/g gallic acid, and/or 0.001-0.1 mg/g trans-caftaric acid,such as 0.005-0.05 mg/g or 0.01-0.025 mg/g trans-caftaric acid.

The aqueous plant extract may be concentrated to a smaller volume, e.g.,by evaporation, and used as an aqueous plant extract. In otherembodiments, the aqueous plant extract is mixed with a carrier beforedrying and grinding. Suitable carriers include, for example,diatomaceous earth, silica, maltodextrin, ground grain (e.g., corn),meals (e.g., soybean or cottonseed meal) by-products (e.g., distiller'sdried grains, rice hulls, wheat mill run), clays (e.g., bentonite), andcombination thereof. The plant extract may be combined with a carrier ina ratio ranging from 10:1 to 1:10 by weight, such as from 5:1 to 1:5.For example, the plant extract may be mixed with diatomaceous earth in aratio of 3:1 by weight.

Additionally, or alternatively, the additional components may comprisecorn, soybean meal, wheat, wheat fiber, barley, rye, rice hulls, canola,limestone, salt, distillers dried grains with solubles (DDGS), dicalciumphosphate, sodium sesquicarbonate, methionine source, lysine source,L-threonine, biotin, folic acid, kelp, menadione dimethylpyrimidinolbisulfite, calcium aluminosilicate, or any combination thereof.

Additional information concerning feed supplement and/or additionalcomponents can be found in PCT application No. PCT/US2015/053439, andU.S. application Ser. Nos. 15/359,342, 14/699,740, 14/606,862, and62/449,959 each of which is incorporated herein by reference in itsentirety.

In some embodiments, the feed supplement does not comprise additionalcomponents. In other embodiments, the feed supplement comprises fromgreater than zero to 40% or more by weight additional components, suchas from 0.1% to 40% by weight, or from 0.2% to 35% by weight additionalcomponents. In certain embodiments, the feed supplement comprises from0.1% to 5% by weight additional components, such as from 0.2% to 3% byweight. In other embodiments, the feed supplement comprises from 5% to20% by weight additional components, such as from 10% to 15% by weight.And in further embodiments, the feed supplement comprises from 20% to40% by weight additional components, such as from 30% to 35% by weightadditional components.

In some embodiments, the feed supplement comprises, consists essentiallyof, or consists of, silica, mineral clay, glucan, mannans, andendoglucanohydrolase; silica, mineral clay, glucan, mannans,endoglucanohydrolase, micro tracers and mineral oil; silica, mineralclay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil,and vitamins; silica, mineral clay, glucan, mannans,endoglucanohydrolase, micro tracers, mineral oil, vitamins, andpotassium sorbate; silica, mineral clay, glucan, mannans,endoglucanohydrolase, vitamins, and active yeast; silica, mineral clay,glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, andactive yeast; silica, mineral clay, glucan, mannans,endoglucanohydrolase, and mineral oil; silica, mineral clay, glucan,mannans, endoglucanohydrolase, vitamins, and calcium carbonate; silica,mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, andwheat fiber; or silica, mineral clay, glucan, mannans,endoglucanohydrolase, and micro tracers. In any of these embodiments,the glucan and mannans may be provided by yeast, yeast cell wall, oryeast cell wall extract.

In some embodiments, the feed supplement does not comprise a peroxidecompound. In some embodiments, the feed supplement does not comprisehydrogen peroxide. In some embodiments, the feed supplement does notcomprise carbamide peroxide. In some embodiments, the feed supplementdoes not comprise urea. In some embodiments, the feed supplement doesnot comprise hydrogen peroxide and urea.

In certain embodiments, the feed supplement is a powdered supplement. Inother embodiments, the feed supplement is a granulated supplement. Thegranulated feed supplement may comprise silica, mineral clay, glucanand/or mannans, and optionally endoglucanohydrolase as discussed above.The granulated feed supplement may have a bulk loose density of from 40lb/ft³ to 150 lb/ft³. In some embodiments, each granule in the granularcomposition comprises silica, mineral clay, glucan and/or mannans, andoptionally endoglucanohydrolase, in relative amounts substantially thesame as a relative amount of each ingredient in the composition aswhole. Each granule in the granular composition may comprise, consistessentially of, or consist of, silica, mineral clay, glucan, mannans andendoglucanohydrolase. Alternatively, or additionally, each granule maycomprise a substantially homogenous blend of silica, mineral clay,glucan and mannans, and optionally endoglucanohydrolase. The compositionmay comprise greater than 40% by weight granules having at least onedimension between 0.149 mm (100 mesh, U.S. standard mesh size) and 4.76mm (4 mesh), and in some embodiments, the composition comprises greaterthan 90% by weight granules having at least one dimension between 0.149mm (100 mesh) and 2 mm (10 mesh). And/or the composition may comprisefrom greater than 0% to 100% granules by weight and from 0% to no morethan 60%, such as no more than 10%, particles by weight, the granuleshaving at least one dimension between 10 mesh (2.00 mm) and 100 mesh(0.149 mm), and the particles having at least one dimension of less than(i.e., smaller than) 100 mesh (0.149 mm). In any embodiments, thegranular composition comprises plural granules, each granule comprisingsilica, mineral clay, glucan and mannans, the granules having a sizethat when administered to an animal increases expression of interleukin10 receptor R (IL10RB) for a time period subsequent to administration,such as subsequent to the onset of administration, relative to an animalthat does not receive the composition. In some embodiments the timeperiod may be from the start of administration to from 28 days to atleast 42 days. And/or the composition may have a mineral coefficient ofvariation of from 0% to 10%, or a proximate coefficient of variation offrom 0% to 20%, or both. Additional information concerning the granularfeed supplement can be found in U.S. application No. 62/449,959 which isincorporated herein by reference in its entirety.

In some embodiments, the feed supplement is administered daily to ananimal at time intervals believed or determined to be effective forachieving a beneficial result. The feed supplement may be administeredin a single dose daily or in divided doses throughout the day. Theamount may be from greater than zero to 500 grams per animal per day,such as from 0.5 grams to 250 grams, from 5 grams to 200 grams, or from10 grams to 70 grams per animal per day. Alternatively, the feedsupplement may be fed or administered in an amount of from greater thanzero to 1000 mgs or more per kilogram of the animal's body weight perday, such as from greater than zero to 500 mgs per kilogram body weight.In other embodiments, the feed supplement is fed or administered perweight of animal feed. The feed supplement may be fed or administered inan amount of from greater than zero to 150 kg per ton (2000 pounds) offeed, such as from 0.1 kg to 100 kg per ton of feed. Alternatively, thefeed supplement may be fed or administered in an amount of from greaterthan zero to 20 grams per kilogram of feed, such as from greater thanzero to 10 grams of feed.

In some embodiments, a composition and/or combination comprises a firstcomposition comprising, consisting essentially of, or consisting of,Bacillus coagulans, Bacillus subtilis, Bacillus licheniformis andBacillus amyloliquefaciens, and a second composition comprising,consisting essentially of, or consisting of, one or more of yucca,quillaja, silica, mineral clay, glucan, mannans, andendoglucanohydrolase. The second composition may comprise, consistessentially of, or consist of, yucca and quillaja, such as Yuccaschidigera and Quillaja saponaria. In other embodiments, the secondcomposition comprises, consists essentially of, or consists of, silica,mineral clay, glucan and mannans, and optionally endoglucanohydrolase.

3. Additional DFM(s)

The disclosed Bacilli combination can be administered to an animal incombination with one or more additional DFMs. The additional DFM(s) maybe any DFM suitable for administration to the particular animal. In someembodiments, the animal is a poultry, particularly a chicken or aturkey, and the additional DFM is a DFM that provides a benefit to thepoultry. The additional DFM may be, by way of example and withoutlimitation, an additional Bacillus species, Lactobacillus, Enterococcus,Bifidobacterium, Propionibacterium, Streptococcus, Pediococcus, yeast,or a combination thereof.

Exemplary additional DFMs include, but are not limited to, Bacillusalcalophilus, Bacillus alvei, Bacillus aminovorans, Bacillusaneurinolyticus, Bacillus anthracis, Bacillus aquaemaris, Bacillusatrophaeus, Bacillus boroniphilus, Bacillus brevis, Bacilluscaldolyticus, Bacillus centrosporus, Bacillus cereus, Bacilluscirculans, Bacillus firmus, Bacillus flavothermus, Bacillus fusiformis,Bacillus galliciensis, Bacillus globigii, Bacillus infernus, Bacilluslarvae, Bacillus laterosporus, Bacillus lentus, Bacillus megaterium,Bacillus mesentericus, Bacillus mucilaginosus, Bacillus mycoides,Bacillus natto, Bacillus pantothenticus, Bacillus polymyxa, Bacilluspseudoanthracis, Bacillus pumilus, Bacillus schlegelii, Bacillussphaericus, Bacillus sporothermodurans, Bacillus stearothermophilus,Bacillus thermoglucosidasius, Bacillus thuringiensis, Bacillus vulgatis,Bacillus weihenstephanensis, Lactobacillus acidophilis, Lactobacillusplantarum, Lactobacillus casei, Lactobacillus gallinarum, Lactobacilluslactis, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillusbulgaricus, Bifidobacterium pseudolongum, Bifidobacterium thermophilium,Bifidobacterium longum, Bifidobacterium lactis, Bifidobacteriumanimalis, Bifidobacterium bifidum, Bifidobacterium infantis,Streptococcus bovis, Streptococcus faecium, Enterococcus faecium,Enterococcus faecalis, Enterococcus diacetylactis, Saccharomycescerevisiae, Saccharomyces boulardii Aspergillus oryzae, Aspergillusniger, Selenomonas ruminantium, Megasphaera elsdenii, Propionibacteriumfreudenreichii, Propionibacterium shermanii, Propionibacteriumacidipropionici, Propionibacterium fensenii, Prevotella bryantii,Pediococcus acidilactici, Pediococcus cerevisiae, or a combinationthereof. In certain embodiments, Bacillus pumilus may be administered incombination with the Bacilli combination.

V. Beneficial Results from Administering the Bacilli Combination

Administering a Bacilli combination to an animal, such as poultry, hasprovided a substantial beneficial result when compared to administeringeach of the respective Bacillus species individually, or in combinationscomprising only two species. These beneficial results are determined byconsidering, for example, feed conversion rate, average body weight,average body weight gain, body weight coefficient of variation, breastmeat yield, bird mortality, lesion scores, necrotic enteritis incidence,Salmonella/E. Coli/Clostridium perfingens (CP) incidence, and/or oocystsin fecal matter at various times during chick rearing. With reference toExample 1, these benefits can be determined by comparing feed conversionrate (FCR) for birds fed a basal diet and challenged with CP relative tobirds that receive the Bacilli combination, such as Bacillus coagulans,Bacillus subtilis and Bacillus licheniformis in combination, or Bacillusamyloliquefaciens, Bacillus subtilis and Bacillus licheniformis,optionally with Bacillus coagulans.

Administration of a disclosed Bacilli combination to poultry may reduceE. coli in the poultry. The amount of E. coli reduction may be fromgreater than zero to 25% or more, such as a reduction of from 5% to 25%,or 10% to 22%, compared to an amount of E. coli present in poultry thatare not administered the combination. The reduction may be identified atvarious times, such as at 21 and/or 42 days old for certain disclosedworking embodiments.

Administration of a disclosed Bacilli combination to poultry may reduceAerobic Plate Count (APC) in the poultry. The APC reduction may be fromgreater than zero to 20% or more, such as a reduction of from 5% to 20%,or 10% to 18%, compared to an amount of APC present in poultry that arenot administered the combination. The reduction may be identified atvarious times, such as at 21 and/or 42 days old for certain disclosedworking embodiments.

Administration of a disclosed Bacilli combination to poultry may reduceSalmonella in the poultry. The Salmonella reduction may be from greaterthan zero to 65% or more, such as a reduction of from 25% to 65%, 35% to65%, or from 45 to 65%, compared to an amount of Salmonella present inpoultry that are not administered the combination. The reduction may beidentified at various times, such as at 21 and/or 42 days old forcertain disclosed working embodiments.

Administration of a disclosed Bacilli combination to poultry may reduceClostridium perfringens in the poultry. The Clostridium perfringensreduction may be from greater than zero to 30% or more, such as areduction of from 5% to 30%, 10% to 30%, or from 15% to 30%, compared toan amount of Clostridium perfringens present in poultry that are notadministered the combination. The reduction may be identified at varioustimes, such as at 21 and/or 42 days old for certain disclosed workingembodiments.

Administration of a disclosed Bacilli combination to poultry may reducefecal oocysts in the poultry. The oocysts reduction may be from greaterthan zero to 90% or more, such as a reduction of from 50% to 90%, or 75%to 90%, compared to an amount of oocysts present in poultry that are notadministered the combination. The reduction may be identified at varioustimes, such as at 21 and/or 42 days old for certain disclosed workingembodiments.

Administration of a disclosed Bacilli combination to poultry may resultin an improved lesion score in the poultry. The lesion score may beimproved (i.e. lowered) by from greater than zero to 75% or more, suchas from 25% to 75%, or from 30% to 75%, compared to a lesion score ofpoultry that are not administered the combination. The improvement maybe identified at various times, such as at 21 and/or 42 days old forcertain disclosed working embodiments.

Administration of a disclosed Bacilli combination to poultry may resultin an improved feed conversion rate in the poultry. The feed conversionrate may be improved (i.e. lowered) by from greater than zero to 10% ormore, such as from 2% to 8%, or from 4% to 8%, compared to a feedconversion rate of poultry that are not administered the combination.The improvement may be identified at various times, such as poultry at14, 21 and/or 42 days old.

Administration of a disclosed Bacilli combination to poultry may resultin a reduced poultry mortality rate. The mortality rate may be reducedby from greater than zero to 95% or more, such as from 50% to 95%, from75% to 95% or from 80% to 95%, compared to a mortality rate of poultrythat are not administered the combination. The improvement may beidentified at various times, such as when the poultry are 14, 21 and/or42 days old.

Administration of a disclosed Bacilli combination to poultry may resultin an improved average weight gain. The weight gain may be increased byfrom greater than zero to 20% or more, such as from 5% to 15%, from 7%to 15% or from 10% to 15%, compared to an average weight gain of poultrythat are not administered the combination. The improvement may beidentified at various times, such as when the poultry are 14, 21 and/or42 days old.

VI. EXAMPLES

The following examples are provided to illustrate certain features ofexemplary working embodiments. A person of ordinary skill in the artwill appreciate that the scope of the invention is not limited to theparticular features of these examples.

Example 1

This example concerns the results of a field study to compare theefficacy of various combinations of direct fed microbials that areadministered in the feed to control necrotic enteritis caused byClostridium perfringens in broiler chickens. The goal of this study iscomparative evaluations of poultry performance in a challenge modelafter administration of different strains or combinations of direct fedmicrobials. The study is performed as a battery trial using a challengemodel wherein the birds are challenged with coccidia and Clostridiumperfringens.

The study consists of 56 cages starting with 8 chicks each. Thetreatments are replicated in 8 blocks of 7 cages each, as shown in Table1.

TABLE 1 Coccidial Clostridium Cages/ Treatment Challenge perfringens TrtT1 Nonmedicated DOT 15 No 8 T2 Nonmedicated DOT 15 DOT 20, 21, and 22 8T3 Gallipro Max 10G, 10 g/t DOT 15 DOT 20, 21, and 22 8 T4 BC1, 7.5 g/tDOT 15 DOT 20, 21, and 22 8 T5 BL1, 113.5 g/t + BS1, 113.5 g/t DOT 15DOT 20, 21, and 22 8 T6 BL1, 113.5 g/t + BS1, 113.5 g/t + DOT 15 DOT 20,21, and 22 8 BC1 6086, 7.5 g/t T7 BL2 + BS2 200 g/t + BC2, 7.5 g/t DOT15 DOT 20, 21, and 22 8

A. Materials and Methods

An un-medicated chicken starter compound is formulated, comprising feedscommonly used in the United States. The diet is representative of alocal commercial formulation and calculated analyses meets or exceedsNRC broiler starter requirements. Experimental treatment feeds areprepared from this basal starter feed. Treatment feeds are mixed toassure a uniform distribution of respective test article. The mixer isflushed to prevent cross contamination. The feed is distributed amongcages of the same treatment. This ration (in mash form) is fed duringthe study.

Day of hatch male broiler chicks are obtained from Cobb-Vantress,Cleveland, Ga. The strain is Cobb 500. Breeder flock information isrecorded. At the hatchery, the birds are sexed and receive routinevaccinations. Only healthy appearing chicks are used in the study.

B. Procedures

The study begins when the birds are placed (day of hatch) (DOT 0) atwhich time they are allocated to experimental cages. No birds arereplaced during the course of the study. All birds are weighed on DOT 0,15, 22, and 28. Feed and water are given ad libitum. Feed is weighed inon DOT 0 and remaining feed is weighed on DOT 15, 22, and 28.

On DOT 15, all birds are orally inoculated with about 5,000 oocysts ofE. maxima. Starting on DOT 20, all birds except Treatment 1 are given abroth culture of C. perfringens about 10⁸ CFU/ml. The birds areadministered a fresh broth culture once daily for 3 days (on DOTs 20,21, and 22). On DOT 22, three birds from each cage are selected,sacrificed, weighed, and examined for the degree of presence of NecroticEnteritis lesions. The scoring is based on a 0 to 3 score, with 0 beingnormal and 3 being the most severe.

The cages are checked twice daily. Observations included are theavailability of feed and water, temperature control, and any unusualconditions. The birds are watched closely for any abnormal reactions.When mortality birds are removed from cages, the cage number, date,weight of the bird, sex, and probable cause of death are recorded. Meansfor cage weight gain, feed consumption, feed conversion, lesion scores,and mortality are calculated.

C. Results

The study is expected to show that administration of the CSL combinationprovides an improved feed conversion rate and/or increased weight gain,compared to birds not administered any Bacillus DFMS, and compared tobirds administered only one or two of the Bacillus species rather thanthe CSL combination.

Additionally, the results will demonstrate that birds that areadministered the CVSL combination have reduced mortality when challengedwith C. perfringens, compared to birds that are challenged but notadministered the CSL combination, such as those not administered aBacillus DFM and those administered only one or two Bacillus species.

Example 2

A field study is performed to compare the efficacy of variouscombinations of direct fed microbials that are administered in the feedto control necrotic enteritis caused by Clostridium perfringens inbroiler chickens. The goal of this study is comparative evaluations ofpoultry performance in a challenge model after administration ofdifferent strains or combinations of direct fed microbials. The study isperformed as a battery trial using a challenge model wherein the birdsare challenged with coccidia and Clostridium perfringens.

The study consisted of 56 cages starting with 8 chicks each. Thetreatments are replicated in 8 blocks of 7 cages each, as shown in Table2.

TABLE 2 Coccidial Clostridium Cages/ Treatment Challenge perfringens TrtT1 Nonmedicated DOT 14 No 8 T2 Nonmedicated DOT 14 DOT 19, 20, and 21 8T3 BC1, 7.5 g/t DOT 14 DOT 19, 20, and 21 8 T4 BC2, 46.875 g/t DOT 14DOT 19, 20, and 21 8 T5 BC2, 7.5 g/t DOT 14 DOT 19, 20, and 21 8 T6 BL1,113.5 g/t BL1 + DOT 14 DOT 19, 20, and 21 8 BS1, 113.5 g/t T7 BL2 + BS2(200 g/t DOT 14 DOT 19, 20, and 21 8

A. Materials and Methods

An un-medicated chicken starter compound is formulated with feedscommonly used in the United States. The diet is representative of alocal commercial formulation and calculated analyses meets or exceedsNRC broiler starter requirements. Experimental treatment feeds areprepared from this basal starter feed. Treatment feeds are mixed toassure a uniform distribution of respective test article. The mixer isflushed to prevent cross contamination. This ration (in mash form) isfed during the study.

Day of hatch male broiler chicks are obtained from Cobb-Vantress,Cleveland, Ga. The strain is Cobb 500. Breeder flock information isrecorded. At the hatchery, the birds are sexed and receive routinevaccinations. Only healthy appearing chicks are used in the study.

B. Procedures

The study begins when the birds are placed (day of hatch) (DOT 0) atwhich time they are allocated to experimental cages. No birds arereplaced during the course of the study. All birds are weighed on DOT 0,14, 21, and 28. Feed is weighed in on DOT 0 and remaining feed isweighed on DOT 14, 21, and 28. Feed and water are given ad libitum.

On DOT 14, all birds are orally inoculated with about 5,000 oocysts ofE. maxima. Starting on DOT 19, all birds except Treatment 1 are given abroth culture of C. perfringens about 10⁸ CFU/ml. The birds areadministered a fresh broth culture once daily for 3 days (on DOTs 19,20, and 21).

On DOT 21, three birds from each cage are selected, sacrificed, weighed,and examined for the degree of presence of Necrotic Enteritis lesions.The scoring is based on a 0 to 3 score, with 0 being normal and 3 beingthe most severe.

The cages are checked twice daily. Observations included are theavailability of feed and water, temperature control, and any unusualconditions. The birds are watched closely for any abnormal reactions.When mortality birds are removed from cages, the cage number, date,weight of the bird, sex, and probable cause of death are recorded. Meansfor cage weight gain, feed consumption, feed conversion, lesion scores,and mortality are calculated.

C. Results

The study is expected to show that administration of the CSL combinationprovides an improved feed conversion rate and/or increased weight gain,compared to birds not administered any Bacillus DFMS, and compared tobirds administered only one or two of the Bacillus species rather thanthe CSL combination.

Additionally, the results will demonstrate that birds that areadministered the CVSL combination have reduced mortality when challengedwith C. perfringens, compared to birds that are challenged but notadministered the CSL combination, such as those not administered aBacillus DFM and those administered only one or two Bacillus species.

Example 3

A total of 4,992 chicks strain were housed at hatch (one day of age, orTrial Day 0) to begin the test feeding period. The chicks were fed asshown in Table 3.

TABLE 3 Ration TEST MATERIAL Number (additives)^(1,2) PHIBRO4-1 Control1 (Clean)-Basal diet + No challenge PHIBRO4-2 Control 2(Challenged)-Basal diet + CP Challenge PHIBRO4-3 Provia 6086 + CPChallenge PHIBRO4-4 Osprey BL + BS + CP Challenge PHIBRO4-5 Osprey BL +BS + Pro via 6086 (recommended levels) + CP Challenge PHIBRO4-6 OspreyBL + BS + Pro via 6086 (@half the recommended levels) + CP ChallengePHIBRO4-7 Control 1 (Clean) + Osprey BL + BS + Pro via 6086 (recommendedlevels) + No Challenge PHIBRO4-8 Calsporin + CP Challenge ¹Eachtreatment was fed to 12 replicates of 52 mixed-sex broilers. ²A basal(with enough feed for the entire treatment) was mixed first and theneach test material was added and remixed. ⁴Recommended levels: Provia6086 (full dose): 7.5 grams per ton of complete feed. Product contains15 billion CFU of Bacillus coagulans per gram resulting in approximately1.2 × 10⁵ CFU per gram in final feed. Osprey BL (full dose): 0.25 lbsper ton of complete feed. Product contains 2.4 × 10⁹ CFU Bacillussubtilis per gram resulting in approximately 3 × 10⁵ CFU per gram infinal feed.

-   -   Osprey BS (full dose): 0.25 lbs per ton of complete feed.        Product contains 2.4×10⁹ CFU Bacillus licheniformis per gram        resulting in approximately 3×10⁵ CFU per gram in final feed.    -   CALSPORIN (N/: 9.07 grams per ton of feed.        The results of this study are summarized in FIG. 1 .

Example 4

This example concerns a field study. The protocol for this study was asshown in Table 4.

TABLE 4 Test Material Test Material (Provia (Osprey 6086 @ BS &+ BL @Ration Treatment 7.5 g/ton of 113.4 g/ton of Number¹ Identification Codefeed)^(2,3) feed, each)^(2,3) PHIBRO7-1 Treatment 1 0 0 (Control, No CP)PHIBRO7-2 Treatment 2 0 0 (Control, CP) PHIBRO7-3 Treatment 3 100% 100%(BL&BS + BC, CP) PHIBRO7-4 Treatment 4  75% 100% (BL&BS + BC, CP)PHIBRO7-5 Treatment 5 100%  75% (BL&BS + BC, CP) PHIBRO7-6 Treatment 6100%  50% (BL&BS + BC, CP) PHIBRO7-7 Treatment 7  75%  50% (BL&BS + BC,CP) PHIBRO7-8 Treatment 8  75%  75% (BL&BS + BC, CP) PHIBRO7-9 Treatment9 0 0 (Calsporin, CP) PHIBRO7-10 Product Y1-1 0 0 (182 g/ton), CPPHIBRO7-11 Product Y2-2 0 0 (182 g/ton), CP ¹Each treatment was fed to12 replicates of 52 mixed-sex broilers. ²A basal feed (with enough feedfor the entire treatment) was mixed first and then each test materialwas added and remixed. ³RECOMMENDED LEVELS: Provia 6086: 7.5 grams perton of complete feed. Product contains 15 billion CFU of Bacilluscoagulans per gram resulting in approximately 1.2 × 10⁵ CFU per gram infinal feed.

Osprey BL (full dose): 0.25 lbs per ton of complete feed. Productcontains 2.4×10⁹ CFU Direct Fed Microbials per gram resulting inapproximately 3×10⁵ CFU per gram in final feed.

Osprey BS (full dose): Use 0.25 lbs per ton of complete feed. Productcontains 2.4×10⁹ CFU Direct Fed Microbials per gram resulting inapproximately 3×10⁵ CFU per gram in final feed.

CALSPORIN (N/C): 9.07 grams per ton of feed.

Product Y1-1 and Y2-2: 182 grams per ton of feed was administered foreach of Treatments #10 and #11.

The results of this study are provided in FIGS. 2 and 3 . With respectto FIGS. 2 and 3 , “Significance (P<0.05)” indicates that averageswithin a row that are without a common superscript are significantlydifferent (P<0.05) as determined by Least Significant Difference.

Example 5

This example concerns the results of a field study to compare theefficacy of various combinations of direct fed microbials that areadministered in the feed to control necrotic enteritis caused byClostridium perfringens in broiler chickens, and the effect of thedirect fed microbials on feed conversion rates and weight gain in thechickens. The goal of this study is comparative evaluations of poultryperformance in a challenge model after administration of differentstrains or combinations of direct fed microbials. The study is performedas a battery trial using a challenge model wherein the birds arechallenged with coccidia and Clostridium perfringens.

The study consists of 56 cages starting with 8 chicks each. Thetreatments are replicated in 8 blocks of 7 cages each, as shown in Table5.

TABLE 5 Coccidial Clostridium Cages/ Treatment Challenge perfringens TrtT1 Nonmedicated DOT 15 No 8 T2 Nonmedicated DOT 15 DOT 20, 21, and 22 8T3 BA DOT 15 DOT 20, 21, and 22 8 T4 BSBL DOT 15 DOT 20, 21, and 22 8 T550%:50% BA:BSBL DOT 15 DOT 20, 21, and 22 8 T6 25%:75% BA:BSBL DOT 15DOT 20, 21, and 22 8 T7 75%:25% BA:BSBL DOT 15 DOT 20, 21, and 22 8BSBL: 1:1 mixture of Bacillus subtilis and Bacillus licheniformis. BA:Bacillus amyloliquefaciens

A. Materials and Methods

An un-medicated chicken starter compound is formulated, comprising feedscommonly used in the United States. The diet is representative of alocal commercial formulation and calculated analyses meets or exceedsNRC broiler starter requirements. Experimental treatment feeds areprepared from this basal starter feed. Treatment feeds are mixed toassure a uniform distribution of respective test article. The mixer isflushed to prevent cross contamination. The feed is distributed amongcages of the same treatment. This ration (in mash form) is fed duringthe study.

Day of hatch male broiler chicks are obtained from Cobb-Vantress,Cleveland, Ga. The strain is Cobb 500. Breeder flock information isrecorded. At the hatchery, the birds are sexed and receive routinevaccinations. Only healthy appearing chicks are used in the study.

B. Procedures

The study begins when the birds are placed (day of hatch) (DOT 0) atwhich time they are allocated to experimental cages. No birds arereplaced during the course of the study. All birds are weighed on DOT 0,15, 22, and 28. Feed and water are given ad libitum. Feed is weighed inon DOT 0 and remaining feed is weighed on DOT 15, 22, and 28.

On DOT 15, all birds are orally inoculated with about 5,000 oocysts ofE. maxima. Starting on DOT 20, all birds except Treatment 1 are given abroth culture of C. perfringens about 10⁸ CFU/ml. The birds areadministered a fresh broth culture once daily for 3 days (on DOTs 20,21, and 22). On DOT 22, three birds from each cage are selected,sacrificed, weighed, and examined for the degree of presence of NecroticEnteritis lesions. The scoring is based on a 0 to 3 score, with 0 beingnormal and 3 being the most severe.

The cages are checked twice daily. Observations included are theavailability of feed and water, temperature control, and any unusualconditions. The birds are watched closely for any abnormal reactions.When mortality birds are removed from cages, the cage number, date,weight of the bird, sex, and probable cause of death are recorded. Meansfor cage weight gain, feed consumption, feed conversion, lesion scores,and mortality are calculated.

C. Results

The study is expected to show that administration of the combination ofBacillus amyloliquefaciens, Bacillus subtilis and Bacillus licheniformisprovides an improved feed conversion rate and/or increased weight gain,compared to birds not administered any Bacillus DFMS, and compared tobirds administered only one or two of the Bacillus species rather thanthe combination of the three Bacilli.

Additionally, the results will demonstrate that birds that areadministered the combination have reduced mortality when challenged withC. perfringens, compared to birds that are challenged but notadministered the combination of Bacillus amyloliquefaciens, Bacillussubtilis and Bacillus licheniformis, such as those not administered aBacillus DFM and those administered only one or two Bacillus species.

Example 6 Study Overview

This study was conducted to determine the dose titration safety andefficacy of Magni-Phi® alone (fed at either 0 and 250 ppm) or incombination with a combination of B. subtilis, B. amyloliquefaciens, B.coagulans and B. licheniformis (administered as Provia Prime™) as wellas other DFM test materials on broiler chicken live performance fromhatch to 42 days of age. Provia Prime™ was fed one level at 0.25 poundsper ton. In particular, Provia Prime™ alone was tested to determine ifmaximum genetic potentials could be met or if feeding both Magni-Phi® atboth levels plus Provia Prime™, as well as other single-straincandidates (B. pumilus, B. subtilis, B. amyloliquefaciens, B. coagulansor B. licheniformis) was beneficial.

The test period began on Trial Day 0 (day of hatch of chicks) and endedon Day 42. The chicks were fed a commercial-type mash feed. The chickswere randomly assigned to one of 15 test groups, which were either 11rep groups (T1-T5) or 4 rep groups (T6-T15) of 52 mixed-sex broilers foreach replication for a total number of 4,940 animals. Chicks wererandomly assigned to treatments on Trial Day 0 (day of hatch) and werenot replaced during the trial. The chicks were observed daily for signsof unusual grow-out patterns or health problems. Body weights and foodconsumption was measured on Trial Days 0, 14, 21, 35 and 42.

Objective, Introduction and Study Overview

1. When broilers are subjected to normal live performance stresses themajor objective is to determine the effect of poultry-related producttest sources from various sources on live performance and processingfactors under typical coccidial-clostridium challenge model stresspoints, when broilers are reared from hatch to 42 days of age in floorpens. The coccidial-clostridium challenge model comprises clostridiumbacteria and coccidiosis challenges, and was administered to all of thebirds within the scope of this trial. The test materials were comparedto both each other, and to a negative control (NC), which was withouttest material administration but included the challenges.

2. The major objective included comparing different combinedpoultry-related product test sources, using levels normally fed in theUSA chicken industry, and determining the effects on broiler chickenmarket weight, mortality and feed conversion on live performance andother test parameters.

3. All birds in this trial were stress challenged by administeringClostridium and coccidia oocysts, along with other natural bacteria frombuild-up litter from a farm experiencing high mortality.

4. Intestinal bacteria were counted, including Clostridium perfringen,E. coli and APC (Aerobic Plate Count), and were measured in Log₁₀counts.

5. Particular to this trial, Salmonella spp. incidence was tested on astatistically sound number of birds (2M and 2F at 21 days of age per52-bird pen and 5M and 5F per replicate at 42 days of age per 52-birdpen) to simulate counts required by USDA/FSIS at processing.

6. Dry Yield (%) and Parts Yield (%), were tested following 42-day bodyweights and live performance measurement.

7. Another objective was to determine if a combination of B. subtilis,B. amyloliquefaciens, B. coagulans and B. licheniformis (administered asProvia Prime™), as well as other Bacillus products, provided an addedbenefit toward ROI (Return-on-Investment) beyond feeding Magni-Phi®alone.

Test System

The commercial-simulated test stress model employed in this study usedbroiler breed chicks (Gallus domesticus) reared on a normal poultryindustry Starter diet (0-21 days of age), Grower diet (22-35 days ofage) and Finisher diet (36-42 days of age) at a floor space requirement(minimum of 0.85 ft² per bird), reared in pens with raised wire floors.Ration formulations (Table 6) were conducted via computer-generatedlinear regression program that simulates formulations conducted duringpractical poultry production techniques. Broilers were fed ad libitumand continuously dosed with their experimental diets from time ofplacement 0 (day of hatch) to up to 42 days of age, as outlined in Table7. Treatments T1-T15 were fed to 11 reps (T1-T5) and 4 reps (T6-T15) of52 mixed-sex broilers. A basal diet (with enough feed for the entiretreatment) was mixed first and then each test material was added andremixed. Each treatments provided a minimum of 5×10⁵ CFU per gram finalfeed. All birds was stressed by administering Clostridium and coccidiaoocysts, along with other natural bacteria from build-up litter from afarm experiencing high mortality.

TABLE 6 Feed Formulation Parameters: Starter Grower Finisher Nutrient0-21 days 22-35 days 36-42 days Metabolizable Energy (kcal/kg) 3,0253,150 3,201 Metabolizable Energy (kcal/#) 1,372 1,429 1,452 Protein (%)22 20 19 Lysine (%) 1.28 1.15 1.02 Methionine (%) Min 0.512 0.46 0.408Methionine + Cysteine (%) 0.947 0.851 0.755 Arginine (%) Min 1.36 1.221.08 Threonine (%) Min 0.858 0.771 0.68 Tryptophan (%) Min 0.19 0.170.15 Total Phosphorus (%) Min 0.70 0.65 0.60 Available Phosphorus (%)0.5 0.45 0.42 Total Calcium (%) 1.05 0.9 0.85 Dietary Sodium (%) 0.200.20 0.20 Dietary Choline (%) 1.35 1.15 0.95

TABLE 7 Experimental Diets Magni-Phi ® TEST Treatment MATERIAL TESTMATERIAL Group (additives) (additives) T1 POSITIVE CLEAN-LITTER NEGATIVECONTROL (PC, No CONTROL (PC, No additive, additive, challenged)challenged) T2 NEGATIVE CHALLENGED NEGATIVE CHALLENGED LITTER LITTERCONTROL (NC, No CONTROL (NC, No additive, additive, challenged)challenged) T3 NC (Trt #2) + Magni-Phi ® None 250 ppm (0.5 lbs. per ton)Alone T4 None NC (Trt #2) + Provia Prime ™ (0.25 Lbs. per ton level). T5NC (Trt #2) + Magni-Phi ® NC (Trt #2) + Provia Prime ™. 250 ppm (0.5lbs. per ton) Alone (0.25 Lbs. per ton level). T6 None NC (Trt #2) + B.subtilis OSPREY (0.25 Lbs. per ton level). T7 NC (Trt #2) + Magni-Phi ®NC (Trt #2) + B. subtilis OSPREY 250 ppm (0.5 lbs. per ton) Alone (0.25Lbs. per ton level). T8 None NC (Trt #2) + B. lichenformis OSPREY (0.25Lbs. per ton level). T9 NC (Trt #2) + Magni-Phi ® NC (Trt #2) + B.lichenformis OSPREY 250 ppm (0.5 lbs. per ton) Alone. (0.25 Lbs. per tonlevel). T10 None NC (Trt #2) + B. coagulans OSPREY (0.25 Lbs. per tonlevel). T11 NC (Trt #2) + Magni-Phi ® NC (Trt #2) + B. coagulans OSPREY250 ppm (0.5 lbs. per ton) Alone. (0.25 Lbs. per ton level). T12 None NC(Trt #2) + B. amyloliquefaciens OSPREY (0.25 Lbs. per ton level). T13 NC(Trt #2) + Magni-Phi ® NC (Trt #2) + B. amyloliquefaciens 250 ppm (0.5lbs. per ton) Alone. OSPREY (0.25 Lbs. per ton level). T14 None NC (Trt#2) + B. pumilus; OSPREY (0.25 Lbs. per ton level). T15 NC (Trt #2) +Magni-Phi ® NC (Trt #2) + B. pumilus; OSPREY 250 ppm (0.5 lbs. per ton)Alone. (0.25 Lbs. per ton level).

Broiler chicks were randomized by individual weight on day of hatch andhoused into each pen onto pens with raised wire floors. Each pen hadsufficient floor, feeder and water space for each growout pen area forhumane treatment of chickens up to 42 days of age. Following 42 days ofgrowout, broilers were weighed, feed consumption determined, lesionscore determined and feed conversion (feed consumed/body weight)calculated.

Materials and Methods Test Material Description

Provia Prime™, a combination of B. subtilis, B. amyloliquefaciens, B.coagulans and B. licheniformis, and Magni-Phi®, which is a compositioncomprising from 10% to 20% Yucca schidigera and from 80% to 90% Quillajasaponaria, were each presented in a premix “powder form”. These were fedat various levels, continuously from Trial Days 0-42, as defined in theexperimental section of this report.

One objective was to compare different test materials, includingMagni-Phi® and Provia Prime™ and other Bacillus species or combinationsthereof, against a Negative Control (providing no other test materialand with a stress challenge) on broiler live performance and lesionscores when broilers were subjected to a sub-clinical challenge(built-up litter and moderate stress conditions via bacterial andcoccidia challenge, when administered). The objective was to determineif Provia Prime™ provided an added benefit toward ROI(Return-on-Investment) beyond feeding Magni-Phi® (250 ppm) or theindividual Bacillus species alone.

Description of Bacteria Challenge (“Test Stress Model”)

The birds were grown on built-up litter sources that containedClostridium perfringens and other pathogenic bacteria, in a“coccidial-clostridium challenge model.” Layout time among flocks was<11 days. Based on previous trials, these organisms were expected to bepresent in the present study. Each pen was top-dressed a minimum of5-million total oocysts (or 100,000-150,000 per bird)+sterile water andnot incorporated into the litter. Coccidia oocysts were predominate E.acervulina and E. maxima. Each pen was “walked with plastic boot coversdesigned to pick up litter” at a minimum of 3-times per day, for thepurpose of spreading the litter from pen-to-pen and equalize thechallenge.

Experimental Design

A total of 5,500 mixed sex broiler chicks (a sufficient number to ensureavailability of at least 4,940 healthy mixed-sex chicks for the conductof the study) were obtained from a commercial hatchery on Trial Day 0(same as hatch date). These were immediately transported to the researchpen units under temperature-controlled conditions to assure birdcomfort. After arrival at the research facility, broilers wereimmediately randomized. There were 52 healthy/viable broiler mixed-sexesper pen with either 11 replicates (Treatments 1-5) or 4 replicates(Treatment 6-15) per test group. Broilers were fed ad libitum theirrespective treatment from day of hatch (termed in this study as TrialDay 0) to 42 days of age.

Broiler Chick Description

Broilers were evaluated upon receipt for signs of disease or othercomplications that may have affected the outcome of the study. Followingexamination, broilers were weighed. Broilers were allocated to each penand to treatment groups using a randomized block design. Weightdistribution across the treatment groups were assessed prior to feedingby comparing the individual test and reference group standard deviationsof the mean against that of the control group. Differences betweencontrol and test or reference groups were within one standard deviation,and as such, weight distribution across treatment groups were consideredacceptable for this study.

Broiler chickens (at hatch to 1-day of age, called Day 0) were collectedin early am (day of placement or Day 0) and randomly assigned to eachexperimental pen within 12 hours of hatch. Weak birds were humanelysacrificed. Birds were not replaced during the study.

Housing and Daily Observations

Each experimental test unit of broiler mixed-sex chicken pens werehoused in separated pens, located in a room containing forced airheaters with a cross-house ventilation system, precision controlled bythe operation manager. Broilers were placed in 5 ft×10 ft pen floor areaand space with a minimum of 0.85 ft² per bird (without feeder andwaterer space) provided. At least two nipple drinkers per pen (via wellwater) provided water.

Two-chick trough feeders per pen were employed for the initial 0-7 daysgrowout period and then larger trough feeders were employed for theremaining growout period.

Continuous (24 hr) use of incandescent lights (LED light system, thatemploys >10 fc) attraction around feeders/waterers for attraction tofeeders/waterers), was used during the entire study. Full lightingof >10 fc (around waterers/feeders only) and >2 fc in remaining floorspace was used the first week and then dimmed to <4 fc for the next twoweeks of age.

Birds were observed at least three times daily for overall health,behavior and/or evidence of toxicity, and environmental conditions.Temperature was checked in the pen room three times daily. Drinkingwater and feed were provided ad libitum.

No type of medication (other than treatment group test material) wasadministered during the entire feeding period. Mortalities wererecorded, refrigerated and, within an 8 hour period, were necropsy (bothinternal and external body mass) by examination examinations wereperformed on all broilers found dead or moribund.

Data and Observations

Live performance body weights and feed intakes were collected on Day 0and 42 during the growing period. Weight gain, feed intake, feed:gainratio (feed efficiency) were calculated for 0-42 days of age. LesionScores were collected at both 21 and 42 days of age. Differences betweenbroilers fed control and test groups were statistically evaluated atP<0.05 in a typical ANOVA analysis of variance test model, employingTreatment x Replicate RCB (Randomized Complete Block). The control groupwas the NC CONTROL with no added test materials.

At the end of the study, all carcasses of necropsied broilers and allbirds remaining at the end of the study were disposed of according tolocal regulations via on-farm composting techniques.

Diet Preparation

Diets were formulated to meet minimum nutrient requirements of a typicalcommercial broiler diet using formulations employed by qualifiednutritionist with training in poultry feed formulations. Actual feedformulations are shown in Table 6, above.

Magni-Phi® and Provia Prime™ feed ingredient sources were added to eachration on an “as is” basis with first mixing small amount of feed withthe basal and then adding the remaining test material to complete themix formula. Dietary protein, lysine, methionine, methionine+cystine,arginine, threonine, tryptophan, total phosphorus, available phosphorus,total calcium, dietary sodium, and dietary choline were met by adjustingthe concentrations of corn and soybean meal ingredients, as well asother minor ingredients commonly used in poultry production. Mixingequipment was flushed with ground corn prior to diet preparation. Alldiets were prepared using a paddle mixer. The mixer was cleaned betweeneach diet using compressed air and vacuum; mixing equipment was flushedwith ground corn between each treatment group and flush material wasretained for disposal.

Diet and Water Administration

Diets were fed in three feed phases: Starter diet (0-21 days of age),Grower diet (22-35 days of age) and Finisher diet (36-42 days of age).All diets were offered ad libitum. Fresh well water (from the researchfacility deep well) was provided ad libitum.

TABLE 8 Test Criteria Performed Data/Sample Collected When Sample SizeMeasurements Feed Intake, BW, Weekly Individual weights Feed Intake, BW,BWG mortality & BW (14, 21, 35 & 42 Adjusted FCR, mortality, BWcoefficient of Uniformity days) variation. Lesion Score Day 21 Day 21 (4birds) 21-day Lesion scores from individual birds and 42 and 42 (10birds (2M and 2F birds). 42-day Lesion scores from per pen) individualbirds (10M and 10F birds). Intestinal Bacteria Days 21 Day 21 (4 birds)Intestinal bacteria levels: Intestinal E. coli, APC and 42 and 42 (10birds (Aerobic Plate Count), Clostridium perfringens per pen) colonieswere enumerated, and Salmonella Incidence determined. Processing DataDays 43 Day 21 (4 birds) Processing Dry Yield (%) to 46 and 42 (10 birdsDry Yield (%) per pen) Breast meat yield (%) Processing Parts Yield (%)Major Parts Yield (%) Breast Meat Yield (% of chilled and live weights)

Results and Discussion

Chicks were randomly assigned to treatments on Trial Day 0 (or day ofhatch). At 42 days of age, live performance (growth weight gain,mortality and feed conversion) and other criteria were determined. Alsoat 42 days of age, lesion scores were measured, and intestinal fecalsamples taken for further laboratory analyses. At 43-46 days of age,processing data were measured.

FIGS. 4-23 provide the data from the study. With respect to dailyobservations, each pen was closely monitored, at a minimum of threetimes per day, to determine overall health, bird behavior and/orevidence of toxicity, and environmental conditions. Temperature (bothhigh and low temperature monitored each time period) was checked withinthe growing area employed for this study three times daily. Temperaturesobserved range from 86-91° F. (Trial Days 0-7), 84-88° F. (Days 8-14),83-85° F. (Days 15-21), 78-83° F. (Days 22-28), 73-77° F. (Days 29-35),and 70-74° F. (Days 36-42).

Mortality in this study for the NC (challenged, no test materials added)ration was considered average to moderate for moderately bacteriabuilt-up litter challenged, by strategy, and on the upper quadrant ofthe Poultry Industry mortality standards (>6% for the NC NegativeControl, with an average mortality in USA is <5%) throughout the growingperiod to 42 days of age, especially for a mild to moderate diseasechallenge model, and caused by bacteria stress (as targeted). Normalpoultry industry mortality is typically and usually <5% when birds aregrown on built-up litter bedding floors, as compared to <9% for a highdisease challenge model. These data indicate that the mild to moderatedisease challenge model functioned as intended.

Significant (P >0.05) improvements in lower mortality rates (i.e., lowermortality) was found with all sources of Magni-Phi® and/or incombination with Provia Prime™, as well as other DFM sources. With theimprovement in mortality with all Magni-Phi® (fed at 250 ppm), ProviaPrime™ and the other DFM sources, these data indicate the safety of theproduct specifically for poultry and livability improvements that may bemade. Furthermore, various significant differences (P >0.05) wereobserved in body weight, feed conversion, and body weight gain (for 0-42days of age) with treatments containing Magni-Phi® (fed at 250 ppm),Provia Prime™ and other DFMs, as compared to the control with no addedtest material but with a stress challenge, that affected liveperformance and mortality.

At 42 days of age, body weights (FIGS. 4-23 ) were significantly(P>0.05) increased and 0-42 day feed conversion significantly (P>0.05)decreased with either Magni-Phi® up to the 250 ppm, as well as otherDFMs and in combination of Magni-Phi® feed additive level. However, whenProvia Prime™ was combined with Magni-Phi® at 250 ppm, the combinationof the two products appeared to statistically (P<0.05) to maximize liveperformance further and beyond just feeding Magni-Phi® alone. In mostlive performance cases in this trial, and with all ages of birds, therewere statistical (P<0.05) improvements in 250 ppm Magni-Phi® vs. NCControl, as well as, feeding the combination of both Magni-Phi® and DFMstest materials, as compared to the Negative Control group of broilerchickens (without either Magni-Phi® or Provia Prime™. Other singleBacillus DFMs showed a similar trend but were of less significance thanwith Provia Prime™

Intestinal bacteria levels (including Intestinal E. coli, APC or AerobicPlate Count, Clostridium perfringens colonies were log₁₀ enumerated andSalmonella Incidence (% of population) was determined at 21 and 42 daysof age. Provia Prime™, in combination with Magni-Phi® fed at 250 ppm,appeared to statistically (P<0.05) maximize live performance, as wellas, intestinal bacteria (Clostridium perfringens, E. coli, APC,Salmonella spp. levels) reductions levels at both 21 and 42 days of age.Additionally, breast meat (either hot carcass weights, chilled weightsand compared as percentage of live weights) showed a similar trendstatistical trend. Intestinal E. coli, Clostridium perfringens andSalmonella Incidence was significantly (P<0.05) reduced with increasinglevels of Magni-Phi®, as well as, the added addition of Provia Prime™ tothese Magni-Phi® levels. The combination of the two test materials ofMagni-Phi® and Provia Prime™, statistically (P<0.05) decreased E. coliand Salmonella incidence at both 250 ppm Magni-Phi® and adding incombination the 1×level of Provia Prime™.

Similar to live performance data (i.e., body weight and feedconversion), intestinal lesion scores on Day 21 and 42 (FIGS. 10 and 11) were significantly (P>0.05) improved with the use of either 250 ppmMagni-Phi®, with or without Provia Prime™.

Similar to live performance data, Dry Yield (%) and Breast Meat Yield (%of chilled and live weights) was statistically (P<0.05) increased withboth Magni-Phi® and Provia Prime™ with the combination of the twoboosting improvements by about >4% for each criterion. Of lesssignificance were changes associated with Processing Parts Yield (%) dueto the additions of either or both Magni-Phi® and Provia Prime™. Othersingle Bacillus DFMs showed a similar trend but were of lesssignificance than with Provia Prime™.

The combination of both Magni-Phi® and Provia Prime™ resulted in mostcriteria from 14-42 days of age being at least numerical, if notstatistically significantly (P<0.05) superior to either Magni-Phi® orProvia Prime™ alone. Additionally, in most cases from 21-42 days of age,the combination of both Magni-Phi® and Provia Prime™ resulted in atleast numerical, ifnot statistically significantly (P<0.05) superior toPC (non-challenged, non-treated Control). Either Magni-Phi® or ProviaPrime™ alone could make this claim. Remarkably, no single-strainBacillus source (including: B. pumilus, B. subtilis,B-amyloliquefaciens, B-coagulans or B-licheniformis) resulted inimprovements over the PC Control ration.

The European Production Efficiency Factor (EPEF) can be sued tostandardize technical results, such as weight gained per day, survivalrate and feed conversion. The EPEF is calculates as follows:

EPEF=(Average grams gained/day×% survival rate)/(Feed Conversion×10).

The EPEF was found statistically (P<0.05) higher (or improved) with thecombination 250 ppm Magni-Phi® and Provia Prime™, although closelybehind individual treatment of either 250 ppm Magni-Phi® and ProviaPrime™, with both appearing to be statistically (P<0.05) similar. Inboth Magni-Phi® addition levels of 250 ppm, Provia Prime™ furtherimproved the genetic potential of the 3-criteria of live body weights,feed conversion and mortality, calculating EPEF, providing the bestperformance combination.

Growth and Feed Conversion graphs (FIGS. 16-23 ) for each test parameterand age group were used to determine potential most efficacious level,via the use of linear regression. In general, the following conclusionswere made, based on these regression analyses curves and graph data:

1. When birds were reared to 21 days of age, maximum mean body weightswere achieved with the combined use of 250 ppm Magni-Phi® and 1X ProviaPrime™. The most efficient feed:gain values were found by feeding atleast 250 ppm Magni-Phi® and Provia Prime™ in combination with 0.25pounds per ton of Provia Prime™.

2. Remarkably, when birds were reared to 42 days of age, statistically(P<0.05) maximum mean body weights were achieved with the combined useof 250 ppm Magni-Phi® plus in combination with Provia Prime™ (at 0.25pounds per ton), as compared to feeding either 250 ppm Magni-Phi® alone,Provia Prime™ alone or NC Control. However, either Magni-Phi® (at the250 ppm Magni-Phi® level) or Provia Prime™ (at 0.25 pounds per ton)levels data were significantly (P<0.05) better than NC Control. In otherwords, the most efficient body weight and feed:gain values were found byfeeding 250 ppm Magni-Phi® in combination with Provia Prime™ followed by250 ppm Magni-Phi® alone, followed by Provia Prime™ alone.

3. With respect to % mortality during the entire trial, statistically(P<0.05) similar mean mortality improvements were achieved with the useof 250 ppm Magni-Phi® in combination with most all DFMs fed in the study(including: Provia Prime™ 4-way DFM combination, B. pumilus, B.subtilis, B-amyloliquefaciens, B-coagulans or B-licheniformis).

4. Both weight gains and feed efficiency values for 22-35 day of ageperiod data were even more dramatically lower for the 250 ppm Magni-Phi®level plus Provia Prime™ level data than the NC Control but wererelatively similar to the PC Control.

5. When birds were reared to 22-35 days or 36-42 days of age periods,higher mean market body weights were achieved with the use of thecombination of Provia Prime™ and 250 ppm Magni-Phi®, as compared toeither 250 ppm Magni-Phi® alone or in combinations with the other DFMssingle strain doses.

6. Similar to both 22-35 and 36-42 days of age results, statistically(P<0.05) improved Body Weight Coefficient of Variation (or CV %) valueswere achieved by feeding Provia Prime™ in combination with 250 ppmMagni-Phi®.

7. Intestinal bacteria content E. coli and APC (Aerobic Plate Counts)were statistically (P<0.05) similar among all test feed articles;however, all treatments were superior to the Negative Control.

8. Intestinal Clostridium perfringens and Salmonella Incidence wassignificantly (P<0.05) reduced with the use of the combination ofMagni-Phi® and Provia Prime™. The combination of the two test materials,statistically (P<0.05) decreased Clostridium perfringens and SalmonellaIncidence levels at 250 ppm Magni-Phi® and were even more dramaticallyreduced by adding in combination the 0.25 pounds per ton Provia Prime™level.

9. Dry Yield (%) and Breast Meat Yield (% of chilled and live weights)was statistically (P<0.05) increased when both Magni-Phi® and ProviaPrime™ were added, with the combination of the two boosting improvementsin processing values.

CONCLUSIONS

Magni-Phi® (fed at 250 ppm) alone or the combination of Provia Prime™(fed at 0.25 pounds per ton level), appeared to improve (at variousefficiency) body weight, feed conversion, mortality, dry yield, breastmeat yield and intestinal bacteria levels at all feed additive additionlevels and combinations, as compared to feeding no ABF feed additive orNEGATIVE CONTROL. The true test in defining essential usage levels is todefine ROI (Return-on-investment) and to determine the“cost-effectiveness” of feeding the combination of products. It appearedthat feeding the combination (as compared to feeding just up to 21 or 35days of age) of all products throughout the entire life-cycle of thebroiler chicken, to 42-days of age and at market age, appeared toimprove live performance and other critical criteria data.

Provia Prime™, as well as all other DFM single strain products, appearedto play a role in aiding Magni-Phi® in improving statistically (P<0.05)maximum live performance, lesion scores and processing characteristics(especially breast meat yield). The most discernible effects ofsignificant improvements (body weight, feed conversion, mortality,breast meat yield and lesion scores at P<0.05) were shown with the useof 250 ppm Magni-Phi® level in combination with Provia Prime™, wheresignificance at the 5% level of probability was found over the NegativeControl Ration (infected and without other test materials), feedingeither product alone, as well as, in most cases including Positive or PCControl Ration (non-infected and without other test materials). Singlestrain DFMs also had an effect, especially when combined withMagni-Phi®.

Remarkably, Magni-Phi®, regardless of DFM usage, appeared toindependently improve growth rate and feed efficiency, all the way tomarket age. Both Magni-Phi® and Provia Prime™ appeared to be feedingredients beneficial to the broiler chicken live performance andprocessing characteristics, but ultimately achieving maximize geneticpotential, as it may relate to cost effectiveness when compared to PCControl or NC Control groups. Provia Prime™ appeared to be an importantcomponent to achieve this very high standard marketing goal, withadvantages over single-strain Bacillus sources ((including: B. pumilus,B. subtilis, B. amyloliquefaciens, B. coagulans or B. licheniformis).

VII. EXEMPLARY EMBODIMENTS

The following numbered statements illustrate exemplary embodiments ofthe disclosed technology.

Statement 1. A Bacilli combination, consisting essentially of three orfour Bacillus species selected from Bacillus coagulans, Bacillussubtilis, Bacillus licheniformis and Bacillus amyloliquefaciens.

Statement 2. A composition, consisting essentially of Bacillus subtilis,Bacillus licheniformis and Bacillus amyloliquefaciens.

Statement 3. The composition of statement 2, consisting of Bacillussubtilis, Bacillus licheniformis and Bacillus amyloliquefaciens.

Statement 4. A composition, consisting essentially of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens and Bacilluscoagulans.

Statement 5. A composition, consisting of Bacillus subtilis, Bacilluslicheniformis, Bacillus amyloliquefaciens and Bacillus coagulans.

Statement 6. A composition, consisting essentially of Bacilluscoagulans, Bacillus subtilis, and Bacillus licheniformis.

Statement 7. The composition of statement 6, consisting of Bacilluscoagulans, Bacillus subtilis, and Bacillus licheniformis.

Statement 8. An admixed composition, comprising:

-   -   a Bacilli combination; and    -   an additional component.

Statement 9. The admixed composition of statement 8, wherein theadditional component comprises a copper salt.

Statement 10. The composition of statement 9, wherein the copper salt iscopper chloride, copper bromide, copper iodide, copper sulfate, coppersulfite, copper bisulfite, copper thiosulfate, copper phosphate,monobasic copper phosphate, dibasic copper phosphate, copperhypophosphite, copper dihydrogen pyrophosphate, copper tetraborate,copper borate, copper carbonate, copper bicarbonate, coppermetasilicate, copper citrate, copper malate, copper methionate, coppersuccinate, copper lactate, copper formate, copper acetate, copperbutyrate, copper propionate, copper benzoate, copper tartrate, copperascorbate, copper gluconate, or a combination thereof.

Statement 11. The admixed composition of statement 8 wherein theadditional component comprises a vitamin.

Statement 12. The admixed composition of any one of statements 5-6 wherethe composition further comprises a vitamin.

Statement 13. The composition of any one of statements 11-12, whereinthe vitamin is vitamin A, vitamin B1, vitamin B₂, vitamin B₃, vitaminB₅, vitamin B₆, vitamin B₁₂, vitamin C, vitamin D, vitamin E, vitamin K,or a combination thereof.

Statement 14. The admixed composition of statement 8 wherein theadditional component comprises an additional direct fed microbial, orstatements 9-13 where the composition further comprises an additionaldirect fed microbial.

Statement 15. The admixed composition of any one of statements 8-14,wherein the Bacilli combination consists essentially of Bacillusamyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis.

Statement 16. The admixed composition of any one of statements 8-14,wherein the Bacilli combination consists essentially of Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus licheniformis andBacillus coagulans.

Statement 17. A composition for administration to poultry, comprising:

-   -   a Bacilli combination; and    -   a poultry feed.

Statement 18. The composition of statement 17, wherein the Bacillicombination consists essentially of Bacillus amyloliquefaciens, Bacillussubtilis, and Bacillus licheniformis.

Statement 19. The composition of statement 17, wherein the Bacillicombination consists essentially of Bacillus amyloliquefaciens, Bacillussubtilis, Bacillus licheniformis and Bacillus coagulans.

Statement 20. The composition of statements 17-19, wherein the poultryfeed comprises plant material, a carbonate, sulfate, lactate, oxide,propionate, stearate, phosphate, mineral, copper species, sugar, salt,animal protein product, forage product, grain product, plant proteinproduct, processed grain product, roughage product, molasses product, orcombinations thereof

Statement 21. The composition of any one of statements 17-20, whereinthe poultry feed comprises beet pulp, ground corn, corn syrup solids,plant fiber, rice hulls, soluble plant fiber, wheat middlings,microcrystalline cellulose, calcium carbonate, potassium carbonate,potassium sulfate, sodium sulfate, calcium lactate, calcium oxide,calcium propionate, calcium stearate, dicalcium phosphate dehydrate,monocalcium phosphate, sodium tripolyphosphate, tetra sodiumpyrophosphate, dolomite, silicon dioxide, silica, limestone,vermiculite, bentonite, montmorillonite, kaolin, glucose, sucrose,dextrose, fructose, maltodextrin, sodium chloride, carrageenan,cellulose, guar gum, polyols, sodium alumino silicate, urea, biotin,folic acid, sodium sesquicarbonate, methionine source, lysine source,L-threonine, or combinations thereof.

Statement 22. The composition of any one of statements 17-21, whereinthe poultry feed comprises copper sulfate.

Statement 23. The composition of any one of statements 17-22, whereinthe composition comprises from 1.2×10⁵ to 4×10⁵ CFU Bacillus subtilisper gram of feed.

Statement 24. The composition of any one of statements 17-23, whereinthe composition comprises from 1.2×10⁵ to 4×10⁵ CFU Bacilluslichemformis per gram of feed.

Statement 25. An admixture composition, comprising:

-   -   a first composition consisting essentially of Bacillus        amyloliquefaciens, Bacillus subtilis, and Bacillus        licheniformis; and    -   a second composition comprising one or more of a feed, yucca,        quillaja, yucca and quillaja, a silica, mineral clay, glucan and        mannans mixture, or a combination thereof.

Statement 26. A composition, comprising Bacillus amyloliquefaciens,Bacillus subtilis, and Bacillus licheniformis and one or more of (a)yucca, quillaja, or yucca and quillaja, (b) silica, mineral clay,glucan, and mannans, or (c) a combination thereof.

Statement 27. A composition formulated for administration to poultry,comprising:

-   -   Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus        licheniformis; and    -   a feed supplement.

Statement 28. A package, comprising:

-   -   a composition consisting essentially of Bacillus        amyloliquefaciens, Bacillus subtilis, and Bacillus        licheniformis; or    -   three separate package portions, one for each of Bacillus        amyloliquefaciens, Bacillus subtilis, and Bacillus        licheniformis.

Statement 29. A method, comprising administering a Bacilli combinationto a subject.

Statement 30. The method according to statement 29 wherein the subjectis livestock.

Statement 31. The method according to statement 24 wherein the Bacillicombination is administered as an admixture comprising a compositionconsisting essentially of Bacillus amyloliquefaciens, Bacillus subtilis,and Bacillus licheniformis and at least one additional component.

Statement 32. A method, comprising administering a compositioncomprising Bacillus amyloliquefaciens, Bacillus subtilis, and Bacilluslicheniformis to poultry.

Statement 33. The method of statement 32, wherein the compositionconsists essentially of Bacillus amyloliquefaciens, Bacillus subtilis,and Bacillus licheniformis.

Statement 34. The method of statement 32, wherein the composition is acomposition according to any one of statements 1-27.

Statement 35. A method, comprising:

-   -   providing Bacillus amyloliquefaciens, Bacillus subtilis, and        Bacillus licheniformis; and    -   forming a first composition comprising Bacillus        amyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis        and a feed, a feed supplement, a direct fed microbial, a        carrier, or a combination thereof.

Statement 36. The method of statement 35, wherein the feed supplementcomprises:

-   -   yucca;    -   quillaja;    -   yucca and quillaja;    -   silica, mineral clay, glucan, and mannans; or    -   a combination thereof.

Statement 37. A composition made by the method of any one of statements35-36.

Statement 38. A method of reducing bird mortality, lesion scores,Salmonella/E. Coli/Clostridium perfingens (CP) incidence, and/or oocystsin fecal matter, comprising administering to poultry an effective amountof a composition comprising Bacillus amyloliquefaciens, Bacillussubtilis, and Bacillus licheniformis.

Statement 39. The method according to statement 38, wherein thecomposition consists essentially of Bacillus amyloliquefaciens, Bacillussubtilis, and Bacillus licheniformis.

Statement 40. A composition for use in administration to poultry,comprising Bacillus amyloliquefaciens, Bacillus subtilis, and Bacilluslicheniformis.

Statement 41. The composition of statement 40 for use in reducing birdmortality, lesion scores, Salmonella/E. Coli/Clostridium perfingens (CP)incidence, and/or oocysts in fecal matter.

Statement 42. The composition of any one of statements 40-41, for use inincreasing breast meat yield.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method, comprising administering to an animal a firstcomposition consisting essentially of from 25% to 50% Bacillus subtilis,from 30% to 65% Bacillus licheniformis, from 5% to 30% Bacillusamyloliquefaciens and from greater than zero to 15% Bacillus coagulans,in amounts relative to each other, wherein the first composition doesnot include any additional direct fed microbial.
 2. The method of claim1, wherein one or more bacillus species in the first composition isdehydrated.
 3. The method of claim 1, wherein the first compositionconsists essentially of from 30% to 45% Bacillus subtilis, from 40% to60% Bacillus licheniformis, from 10% to 25% Bacillus amyloliquefaciensand from 1% to 12% Bacillus coagulans.
 4. The method of claim 1, whereinthe animal is livestock.
 5. The method of claim 1, wherein the animal ispoultry.
 6. The method of claim 1, wherein administration of thecomposition increases breast meat yield, increases weight gain, improvesa feed conversion ratio, reduces bird mortality, reduces lesion scores,reduces Salmonella/E. Coli/Clostridium perfingens (CP) incidence,reduces oocysts in fecal matter, or a combination thereof.
 7. The methodof claim 1, wherein the first composition further includes a carrier. 8.The method of claim 1, wherein the method further comprisesadministering an additional component.
 9. The method of claim 8, whereinthe additional component is administered simultaneously with the firstcomposition.
 10. The method of claim 8, wherein the additional componentcomprises yucca, quillaja, silica, mineral clay, glucan, mannans,endoglucanohydrolase, a copper salt, a vitamin, or a combinationthereof.
 11. The method of claim 8, wherein the additional componentcomprises Yucca schidigera, Quillaja saponaria, or a combinationthereof.
 12. The method of claim 8, wherein the additional componentcomprises a second composition comprising silica, mineral clay, glucan,mannans and endoglucanohydrolase.
 13. The method of claim 8, whereinadministering the first composition comprises administering a feedcomposition comprising the first composition and an animal feed.
 14. Themethod of claim 13, wherein the animal feed is a poultry feed.
 15. Themethod of claim 13, wherein the feed composition comprises from 1.2×10⁵to 4×10⁵ CFU Bacillus subtilis per gram of animal feed.
 16. The methodof claim 13, wherein the feed composition comprises from 1.2×10⁵ to4×10⁵ CFU Bacillus licheniformis per gram of animal feed.
 17. The methodof claim 13, wherein the feed composition comprises from 0.1 pound to 1pound of the first composition per ton of animal feed.
 18. The method ofclaim 13, wherein the feed composition further comprises from 100 ppm to500 ppm of a second composition comprising Yucca schidigera and Quillajasaponaria, per ton of animal feed.
 19. A method of reducing birdmortality, lesion scores, Salmonella/E. Coli/Clostridium perfingens (CP)incidence, and/or oocysts in fecal matter, comprising administering topoultry an effective amount of a composition consisting essentially offrom 25% to 50% Bacillus subtilis, from 30% to 65% Bacilluslicheniformis, from 5% to 30% Bacillus amyloliquefaciens and fromgreater than zero to 15% Bacillus coagulans, in amounts relative to eachother, wherein the composition does not include any additional directfed microbial.
 20. The method of claim 19, wherein the method furthercomprises administering Yucca schidigera and Quillaja saponaria to thepoultry.